CN112955557A - Gene therapy for treating propionic acidemia - Google Patents

Abstract

The present disclosure provides adeno-associated viral vectors, recombinant adeno-associated viruses (rAAV), and methods of their use in gene therapy for the treatment of Propionemia (PA). Also provided is a pharmaceutical composition comprising the recombinant adeno-associated virus of the invention and a pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions may be useful in gene therapy for the treatment of PA caused by a mutation in propionyl-coa carboxylase alpha-subunit (PCCA) or a mutation in propionyl-coa carboxylase beta-subunit (PCCB).

Description

Gene therapy for treating propionic acidemia

Cross reference to related applications

This application claims benefit and priority to U.S. provisional patent application No. 62/739,471 filed on 1/10/2018, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

Sequence listing

This application contains a sequence listing submitted electronically in ASCII format and incorporated by reference herein in its entirety. The ASCII copy generated in 9, and 22 days in 2019 is named ULP-001WO _ SL _ ST25.txt and has the size of 98,889 bytes.

Technical Field

The present disclosure relates generally to adeno-associated viral vectors, adeno-associated viruses, and methods of their use in gene therapy for the treatment of propionic acid blood disease.

Background

Propionemia (PA), also known as propionic urine, is a congenital error in the metabolism of organic acids caused by the absence of active propionyl-coa carboxylase (PCC), an enzyme required for the conversion of propionyl-coa to (D) -methylmalonyl-coa, a key step in the catabolic pathways of odd-chain fatty acids and propionic acid producing amino acids, in particular isoleucine, threonine, methionine and valine. PCC enzymes consist of two distinct subunits, alpha and beta, which are encoded by the PCCA and PCCB genes, respectively. Propionyl-coa carboxylase defects may be caused by mutations in either PCCA or PCCB. In one study, 30 PA patients were subjected to mutation analysis and 15 patients were found to have α -subunit deficiency and 15 patients had β -subunit deficiency. See Yang et al, 2004, Mol Genet and Metab.81: 335-.

The estimated incidence of PA in the united states is 1:105,000 to 1:130,000. This rare autosomal recessive metabolic disorder manifests itself in early neonatal onset as poor eating, vomiting, lethargy, seizures and lack of muscle tone. If left untreated, death may occur rapidly due to secondary hyperammonemia, infection, cardiomyopathy, or basal segment stroke. PA can be diagnosed almost immediately in neonates, and the disease is included in the neonatal screening group of diseases in the united states.

Currently, PA is regulated by dietary restriction of amino acid precursors, supplementation of L-carnitine to solve the problem of carnitine level reduction, and administration of antibiotics to reduce propionic acid production by intestinal bacteria. Liver transplantation plays a role in the management of PA in cases where patients cannot be managed by standard therapy. However, despite active attempts to address the disease through complex combinations of nutrition, cofactors and antibiotic therapy, the long-term prognosis of PA patients remains poor. See van der Meer et al, 1996, Eur.J.Pediatr.155: 205-210. Thus, there is a need for improved treatments that address the underlying cause of the disease, namely the lack of PCC.

The present invention addresses this need by generating adeno-associated viral vectors that mediate the transfer of functional PCCA or PCCB genes to PA patients. The invention also describes the production of recombinant adeno-associated virus (rAAV) that delivers functional PCCA or PCCB genes to PA patients.

Disclosure of Invention

The present invention provides compositions and methods of their use in gene therapy. More specifically, provided herein are recombinant adeno-associated viruses (rAAV) comprising an adeno-associated virus (AAV) capsid and a vector genome packaged therein that are useful for treating PA.

In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, in 5 'to 3' order: (a) a5 '-inverted terminal repeat (5' -ITR) sequence; (b) a promoter sequence; (c) a partial or complete coding sequence for PCCA; and (d) a 3 '-inverted terminal repeat (3' -ITR) sequence.

In another aspect, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, in 5 'to 3' order as operably linked components: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) a partial or complete coding sequence for PCCA; (f) a polyadenylation signal sequence; and (g) a 3' -ITR sequence.

In certain embodiments, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising AAV5 '-ITR sequences, promoter sequences, partial or complete coding sequences of PCCA or an isoform or functional fragment or functional variant thereof, and AAV 3' -ITR sequences.

In certain embodiments, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of a human PCCA 5' -untranslated region (5 '-UTR), a partial or complete coding sequence of PCCA or an isoform or functional fragment or functional variant thereof, a truncated or complete nucleotide sequence of a human PCCA 3' -untranslated region (3 '-UTR), and an AAV 3' -ITR.

In certain embodiments, the present disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising a nucleic acid sequence consisting of SEQ ID NO: 30, intact human PCCA 5' -UTR. In certain embodiments, the packaged genome comprises a truncated human PCCA 5' -UTR nucleotide sequence. In certain embodiments, the truncated nucleotide sequence of the human PCCA 5' -UTR comprises SEQ ID NO: 30 of the container. In certain embodiments, the truncated nucleotide sequence of the human PCCA 5' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 30, and a nucleotide sequence of at least 100 contiguous nucleotides with at least 95% identity over a region of equal length. In certain embodiments, when part or all of the coding sequence in the packaged vector genome is for PCCA, the truncated or all of the human PCCA 5' -UTR is located between the intron sequence and part or all of the coding sequence of PCCA.

In certain embodiments, the packaged vector genome comprises a nucleic acid sequence consisting of SEQ ID NO: 31, the complete human PCCA 3' -UTR. In certain embodiments, the packaged genome comprises a truncated human PCCA 3' -UTR nucleotide sequence. In certain embodiments, the truncated nucleotide sequence of the human PCCA 3' -UTR comprises SEQ ID NO: 31. In certain embodiments, the truncated nucleotide sequence of the human PCCA 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 31, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length. In certain embodiments, when part or all of the coding sequence in the packaged vector genome is for PCCA, the truncated or all of the human PCCA 3 '-UTR is located between the polyadenylation signal sequence and the 3' -ITR sequence.

In one embodiment, the partial or complete coding sequence of PCCA is a wild-type coding sequence. In alternative embodiments, the partial or complete coding sequence of PCCA is a codon optimized coding sequence. In an exemplary embodiment, the partial or complete coding sequence of PCCA is codon optimized for expression in humans.

In certain embodiments, the PCCA consists of SEQ ID NO: 1, or a wild-type coding sequence shown in seq id no. In another embodiment, coding sequences expressing native isoforms or variants of PCCA may be used, such as shown in UniProtKB/Swiss-Prot accession numbers P05165-1(SEQ ID NO: 25), P05165-2(SEQ ID NO: 26), and P05165-3(SEQ ID NO: 27). In certain embodiments, the PCCA is encoded by a codon-optimized coding sequence.

In certain embodiments, the PCCA consists of a sequence identical to SEQ ID NO: 1 has less than 80% identity to the wild-type coding sequence. In certain exemplary embodiments, the PCCA consists of a nucleotide sequence selected from SEQ ID NOs: 2-6. In certain embodiments, the PCCA consists of a nucleotide sequence identical to a nucleotide sequence selected from SEQ ID NOs: 2-6, a codon optimized coding sequence that is at least 80% identical in sequence. In certain embodiments, the PCCA consists of a nucleotide sequence identical to a nucleotide sequence selected from SEQ ID NOs: 2-6, a codon optimized coding sequence that is at least 90% identical in sequence. In certain embodiments, the PCCA consists of a nucleotide sequence identical to a nucleotide sequence selected from SEQ ID NOs: 2-6, a codon optimized coding sequence that is at least 95% identical in sequence. In certain embodiments, the coding sequence for PCCA may further comprise a stop codon (TGA, TAA or TAG) at the 3' terminus. In certain embodiments, the expressed PCCA comprises SEQ ID NO: 16 or consists thereof.

In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, in 5 'to 3' order: (a) 5' -ITR sequence; (b) a promoter sequence; (c) a partial or complete coding sequence of PCCB; and (d) a 3' -ITR sequence.

In another aspect, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, in 5 'to 3' order as operably linked components: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) a partial or complete coding sequence of PCCB; (f) a polyadenylation signal sequence; and (g) a 3' -ITR sequence.

In certain embodiments, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising AAV5 '-ITR sequences, promoter sequences, partial or complete coding sequences of PCCB or isoforms or functional fragments or functional variants thereof, and AAV 3' -ITR sequences.

In certain embodiments, the disclosure provides a rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of a human PCCB 5' -untranslated region (5 '-UTR), a partial or complete coding sequence of a PCCB or an isoform or functional fragment or functional variant thereof, a truncated or complete nucleotide sequence of a human PCCB 3' -untranslated region (3 '-UTR), and an AAV 3' -ITR.

In certain embodiments, the packaged vector genome comprises a nucleic acid sequence consisting of SEQ ID NO: 32, complete human PCCB 5' -UTR. In certain embodiments, the packaged vector genome comprises a truncated human PCCB 5' -UTR nucleotide sequence. In certain embodiments, the truncated nucleotide sequence of the human PCCB 5' -UTR comprises SEQ ID NO: 32. In certain embodiments, the truncated nucleotide sequence of the human PCCB 5' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 32, and a nucleotide sequence of at least 100 contiguous nucleotides that are at least 95% identical over a region of equal length. In certain embodiments, when the partial or complete coding sequence in the packaged vector genome is for PCCB, the truncated or complete human PCCB 5' -UTR is located between the intron sequence and the partial or complete coding sequence of PCCB.

In certain embodiments, the packaged vector genome comprises a nucleic acid sequence consisting of SEQ ID NO: 33, complete human PCCB 3' -UTR. In certain embodiments, the packaged vector genome comprises a truncated human PCCB 3' -UTR nucleotide sequence. In certain embodiments, the truncated nucleotide sequence of the human PCCB 3' -UTR comprises SEQ ID NO: 33. In certain embodiments, the truncated nucleotide sequence of the human PCCB 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 33, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length. In certain embodiments, when part or all of the coding sequence in the packaged vector genome is for PCCB, the truncated or all of the human PCCB 3 '-UTR is located between the polyadenylation signal sequence and the 3' -ITR sequence.

In one embodiment, the partial or complete coding sequence of the PCCB is a wild-type coding sequence. In alternative embodiments, the partial or complete coding sequence of the PCCB is a codon optimized coding sequence. In an exemplary embodiment, the partial or complete coding sequence of the PCCB is codon optimized for expression in humans.

In certain embodiments, the PCCB consists of SEQ ID NO: 7, or a wild-type coding sequence shown in seq id no. In another embodiment, coding sequences expressing native isoforms or variants of PCCB may be used, such as shown in UniProtKB/Swiss-Prot accession numbers P05166-1(SEQ ID NO: 28) and P05166-2(SEQ ID NO: 29). In alternative embodiments, the PCCB is encoded by a codon optimized coding sequence. In certain embodiments, the PCCB is formed from a nucleic acid sequence identical to SEQ ID NO: 7 has less than 80% identity to the wild type coding sequence. In certain exemplary embodiments, the PCCB is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8-12. In certain embodiments, the PCCB is formed from a nucleic acid sequence identical to a sequence selected from SEQ ID NOs: 8-12 is encoded by a codon-optimized coding sequence that is at least 80% identical in sequence. In certain embodiments, the PCCB is formed from a nucleic acid sequence identical to a sequence selected from SEQ ID NOs: 8-12 is at least 90% identical to the sequence of the codon optimized coding sequence coding. In certain embodiments, the PCCB is formed from a nucleic acid sequence identical to a sequence selected from SEQ ID NOs: 8-12 is at least 95% identical to the sequence of the codon optimized coding sequence coding. In certain embodiments, the coding sequence of the PCCB may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus. In certain embodiments, the expressed PCCB comprises SEQ ID NO: 17 or consists thereof.

In certain embodiments, the 5' -ITR sequence is from AAV 2. In certain embodiments, the 3' -ITR sequence is from AAV 2. In certain embodiments, the 5 '-ITR and 3' -ITR sequences are from AAV 2. In certain embodiments, the 5 '-ITR sequence and/or the 3' -ITR sequence comprises SEQ ID NO: 15 or consist thereof. In other embodiments, the 5 '-ITR and/or 3' -ITR sequences are from a non-AAV 2 source.

In one embodiment, the promoter is selected from the group consisting of chicken β -actin (CBA) promoter, Cytomegalovirus (CMV) immediate early gene promoter, transthyretin (TTR) promoter, thyroxine-binding globulin (TBG) promoter, alpha-1 antitrypsin factor (A1AT) promoter, CAG promoter (constructed using CMV early enhancer element, promoter, first exon and first intron of CBA gene, and splice acceptor of rabbit β -globin gene), PCCA gene-specific endogenous promoter, and PCCB gene-specific endogenous promoter. In an exemplary embodiment, the promoter is a CBA promoter. In one embodiment, the CBA promoter comprises SEQ ID NO: 18 or consist thereof. In certain embodiments, the promoter is a gene-specific endogenous promoter. In one embodiment, the promoter comprises an endogenous gene promoter element. In an exemplary embodiment, when part or the entire coding sequence in the vector genome is for PCCA, the promoter is a PCCA gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 34, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length. In an exemplary embodiment, when part or the entire coding sequence in the vector genome is for PCCB, the promoter is a PCCB gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 36, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length.

In certain embodiments, the packaged vector genome further comprises one or more enhancer sequences. In one embodiment, the enhancer is selected from the Cytomegalovirus (CMV) immediate early gene enhancer, the transthyretin enhancer (enTTR), the chicken β -actin (CBA) enhancer, the En34 enhancer, and the apolipoprotein e (apoe) enhancer. In an exemplary embodiment, the enhancer is a CMV enhancer. In one embodiment, the CMV enhancer comprises SEQ ID NO: 19 or consist thereof. In certain embodiments, the enhancer is located upstream of the promoter sequence.

In certain embodiments, the packaged vector genome further comprises one or more intron sequences. In one embodiment, the intron is selected from the group consisting of the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the beta globin/IgG chimeric intron (Promega chimeric intron), and the hFIX intron. In an exemplary embodiment, the intron is the SV40 small T intron. In one embodiment, the SV40 small T intron sequence comprises SEQ ID NO: 20 or consist thereof. In another exemplary embodiment, the intron is a rHBB intron. In one embodiment, the rHBB intron sequence comprises SEQ ID NO: 21 or consist thereof.

In certain embodiments, the packaged vector genome further comprises a consensus Kozak sequence. In certain embodiments, the consensus Kozak sequence is located downstream of an intron sequence. In one embodiment, the consensus Kozak sequence is GCCGCC (SEQ ID NO: 24). In certain embodiments, the consensus Kozak sequence is located upstream of the coding sequence of PCCA. In certain embodiments, the consensus Kozak sequence is located upstream of the coding sequence of PCCB.

In certain embodiments, the packaged vector genome further comprises a polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence is selected from the group consisting of a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, a rabbit β globin polyadenylation signal sequence, a PCCA gene-specific endogenous polyadenylation signal sequence, a PCCB gene-specific endogenous polyadenylation signal sequence. In an exemplary embodiment, the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence. In one embodiment, the BGH polyadenylation signal sequence comprises SEQ ID NO: 22 or consist thereof. In another exemplary embodiment, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence. In one embodiment, the SV40 polyadenylation signal sequence comprises SEQ ID NO: 23 or consist thereof. In another exemplary embodiment, when part or all of the coding sequence in the vector genome is for PCCA, the polyadenylation signal sequence comprises a PCCA gene-specific endogenous polyadenylation signal sequence. In one embodiment, the PCCA gene-specific endogenous polyadenylation signal sequence comprises a sequence identical to SEQ ID NO: 35, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length. In another exemplary embodiment, when part or all of the coding sequence in the vector genome is for PCCB, the polyadenylation signal sequence comprises a PCCB gene-specific endogenous polyadenylation signal sequence. In one embodiment, the PCCB gene-specific endogenous polyadenylation signal sequence comprises a nucleotide sequence identical to SEQ ID NO: 37, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

In certain embodiments, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10 or hu 37. In an exemplary embodiment, the AAV capsid is from AAV 9. In another exemplary embodiment, the AAV capsid is from AAV 8. In another exemplary embodiment, the AAV capsid is an AAV9 variant capsid.

In certain embodiments, the disclosure provides a rAAV, wherein the coding sequence is for PCCA but not PCCB, the promoter is a PCCA gene-specific endogenous promoter but not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence but not a PCCB gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the disclosure provides a rAAV, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence.

In certain instances, the present disclosure provides novel codon-optimized nucleic acid sequences encoding PCCA. In one embodiment, the PCCA-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NO: 1 has less than 80% identity to the wild type coding sequence shown in figure 1. In certain embodiments, the PCCA-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NOs: 2-6 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In certain embodiments, the present disclosure provides a polypeptide that differs from SEQ ID NO: 1 and has less than 80% identity to the wild type coding sequence shown in SEQ ID NOs: 2-6 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In exemplary embodiments, the present disclosure provides a polypeptide selected from SEQ ID NOs: 2-6 encoding PCCA. Also provided are SEQ ID NOs: 2-6 encoding a polypeptide having functional PCCA activity. In certain embodiments, the nucleic acid sequence encoding PCCA may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus.

In certain instances, the present disclosure provides novel codon-optimized nucleic acid sequences encoding PCCBs. In one embodiment, the PCCB-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NO: 7 have less than 80% identity. In certain embodiments, the PCCB-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NOs: 8-12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. The present disclosure provides methods and compositions similar to SEQ ID NO: 7 and has less than 80% identity to the wild type coding sequence shown in SEQ ID NOs: 8-12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In exemplary embodiments, the present disclosure provides a polypeptide selected from SEQ ID NOs: 8-12 encoding a PCCB. Also provided are SEQ ID NOs: 8-12 encoding a polypeptide having functional PCCB activity. In certain embodiments, the nucleic acid sequence encoding PCCB may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus.

In certain embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) useful as a gene therapy agent for treating PA, wherein the rAAV comprises an AAV capsid and a vector genome described herein packaged therein. In certain embodiments, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10 or hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAVrh10, or AAVhu 37). In exemplary embodiments, the AAV vector is an AAV serotype 9(AAV9) vector, an AAV9 variant vector, an AAV serotype 8(AAV8) vector, or an AAV serotype 2(AAV2) vector.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA; (f) a polyadenylation signal sequence; and (g) a 3' ITR sequence.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB; (f) a polyadenylation signal sequence; and (g) a 3' ITR sequence.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA; (f) a polyadenylation signal sequence; and (g) a 3' -ITR sequence.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) 5' -ITR sequence; (b) an enhancer sequence; (c) a promoter sequence; (d) an intron sequence; (e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB; (f) a polyadenylation signal sequence; and (g) a 3' -ITR sequence.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) AAV 25' -ITR sequences; (b) a CMV enhancer sequence; (c) a CBA promoter sequence; (d) rHBB or SV40 small T intron sequences; (e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA; (f) a BGH or SV40 polyadenylation signal sequence; and (g) AAV 23' -ITR sequences.

In certain embodiments, the present disclosure provides a rAAV useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order: (a) AAV 25' -ITR sequences; (b) a CMV enhancer sequence; (c) a CBA promoter sequence; (d) rHBB or SV40 small T intron sequences; (e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB; (f) a BGH or SV40 polyadenylation signal sequence; and (g) AAV 23' -ITR sequences.

In certain embodiments, the present disclosure provides a recombinant nucleic acid construct comprising (a) a 5' -ITR sequence; (b) a promoter sequence; (c) a partial or complete coding sequence for PCCA; and (d) a 3' -ITR sequence. In certain embodiments, the present disclosure provides a recombinant nucleic acid construct comprising (a) a 5' -ITR sequence; (b) a promoter sequence; (c) a partial or complete coding sequence of PCCB; and (d) a 3' -ITR sequence.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating PA, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In certain embodiments, the rAAV contains a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, a promoter sequence, a partial or complete coding sequence of PCCA or its isoform or a functional fragment or functional variant thereof, and a 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3' -ITR. In one exemplary embodiment, the rAAV contains a packaged genome comprising, in 5 'to 3' order as operably linked components: AAV 25 '-ITR sequences, CMV enhancer, CBA promoter, SV40 small T intron, coding sequence for PCCA, BGH polyadenylation signal sequence and AAV 23' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In certain embodiments, the PCCA coding sequence is selected from SEQ ID NOs: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating PA, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In certain embodiments, the packaged genome comprises, in 5 'to 3' order as operably linked components: 5 '-ITR, a promoter sequence (e.g., a PCCA gene-specific endogenous promoter sequence), a truncated or complete nucleotide sequence of the human PCCA 5' -UTR, a partial or complete coding sequence of PCCA or its isoform or functional fragment or functional variant thereof, a truncated or complete nucleotide sequence of the human PCCA 3 '-UTR, and a 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a PCCA gene-specific polyadenylation sequence upstream of the 3' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the truncated or complete nucleotide sequence of the human PCCA 5' -UTR and upstream of a portion or the complete coding sequence of PCCA or its isoform or functional fragment or functional variant thereof. In certain embodiments, the PCCA coding sequence is selected from SEQ ID NOs: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating PA, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In certain embodiments, the packaged genome comprises, in 5 'to 3' order as operably linked components: 5 '-ITR, a promoter sequence, a partial or complete coding sequence of PCCB or its isoform or a functional fragment or functional variant thereof, and a 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3' -ITR. In an exemplary embodiment, the packaged genome comprises as operably linked components, in 5 'to 3' order: AAV 25 '-ITR sequences, CMV enhancer, CBA promoter, SV40 small T intron, coding sequence for PCCB, BGH polyadenylation signal sequence and AAV 23' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In certain embodiments, the coding sequence of the PCCB is selected from the group consisting of SEQ ID NOs: 7-12. In certain embodiments, the capsid is an AAV9 capsid.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating PA, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In certain embodiments, the packaged genome comprises, in 5 'to 3' order as operably linked components: 5 '-ITR, promoter sequences (e.g., PCCB gene specific endogenous promoter sequences), truncated or complete nucleotide sequences of human PCCB 5' -UTR, partial or complete coding sequences of PCCB or an isoform or functional fragment or functional variant thereof, truncated or complete nucleotide sequences of human PCCB 3 '-UTR, and 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a PCCB gene-specific polyadenylation sequence upstream of the 3' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the truncated or complete nucleotide sequence of the human PCCB 5' -UTR and upstream of a part or the complete coding sequence of PCCB or an isoform or a functional fragment or functional variant thereof. In certain embodiments, the coding sequence of the PCCB is selected from SEQ ID NO: 7-12. In certain embodiments, the capsid is an AAV9 capsid.

The present disclosure also relates to pharmaceutical compositions comprising the rAAV disclosed herein. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In exemplary embodiments, the pharmaceutical composition is formulated for intravenous administration.

In another aspect, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to the human subject a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCA or an isoform or a functional fragment or variant thereof. In another embodiment, the present disclosure provides a method of treating PA comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCB or an isoform or a functional fragment or variant thereof.

In certain embodiments, the present disclosure provides a method of treating PA, the method comprising administering: (1) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCA or an isoform or a functional fragment or variant thereof; and (2) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence of PCCB or an isoform or a functional fragment or functional variant thereof. In certain embodiments, the rAAV of (1) and (2) may be administered simultaneously. In certain embodiments, the rAAV of (1) and (2) may be administered sequentially. In certain embodiments, the rAAV of (1) and (2) may be administered separately.

In certain embodiments, the present disclosure provides a method of treating PA, the method comprising administering: (1) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCA and not PCCB, the promoter is a PCCA gene-specific endogenous promoter and not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence and not a PCCB gene-specific endogenous polyadenylation signal sequence; and (2) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the rAAV of (1) and (2) may be administered simultaneously. In certain embodiments, the rAAV of (1) and (2) may be administered sequentially. In certain embodiments, the rAAV of (1) and (2) may be administered separately.

In certain embodiments, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to a human subject diagnosed as having at least one mutation in PCCA a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCA or an isoform or functional fragment or functional variant thereof. In certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCA but not PCCB, the promoter is a PCCA gene-specific endogenous promoter but not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence but not a PCCB gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the mutation in PCCA is selected from table 1. In certain embodiments, the PCCA coding sequence is selected from SEQ ID NOs: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

In certain instances, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to a human subject diagnosed as having at least one mutation in PCCB a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCB or an isoform or functional fragment or functional variant thereof. In certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the mutation in the PCCB is selected from table 2. In certain embodiments, the coding sequence of the PCCB is selected from the group consisting of SEQ ID NOs: 7-12. In certain embodiments, the capsid is an AAV9 capsid.

In certain embodiments, the rAAV is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, or intravenously. In an exemplary embodiment, the rAAV is administered intravenously. In some embodiments of the present invention, the substrate is,the rAAV is about 1x10¹¹To about 1x10¹⁴Genomic Copy (GC)/kg. In other embodiments, the rAAV is administered at about 1x10¹²To about 1x10¹³Genomic Copy (GC)/kg. In certain embodiments, a single dose of rAAV is administered. In other embodiments, multiple doses of rAAV are administered.

In certain instances, provided herein are host cells comprising a recombinant nucleic acid molecule, AAV vector, or rAAV disclosed herein. In particular embodiments, the host cell may be suitable for propagation of AAV. In certain embodiments, the host cell is selected from HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19 or MRC-5 cells.

These and other aspects and features of the present invention are described in the following sections of this disclosure.

Drawings

The present invention may be more completely understood with reference to the following drawings.

FIG. 1 is a schematic diagram illustrating an exemplary packaged vector genomic construct comprising PCCA, according to one embodiment. In the figure, the 5 '-ITR, CMV enhancer, chicken β -actin promoter, SV40 small T intron, consensus Kozak sequence, PCCA coding sequence, SV40 polyadenylation signal, and 3' -ITR are represented by 1, 2, 3, 4, 5, 6, 7, and 8, respectively.

FIG. 2 is a schematic diagram illustrating an exemplary packaged vector genomic construct comprising PCCB, according to one embodiment. In the figure, the 5 '-ITR, CMV enhancer, chicken β -actin promoter, SV40 small T intron, consensus Kozak sequence, PCCB coding sequence, SV40 polyadenylation signal, and 3' -ITR are represented by 1, 2, 3, 4, 5, 6, 7, and 8, respectively.

FIG. 3 is a bar graph showing the fold change in PCCA RNA expression determined by RT-qPCR after transfection of continuous liver cell lines (HepG2) with control plasmids (plasmids with empty vector, without TX), a rAAV vector plasmid with a nucleotide sequence encoding the enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the entire native sequence of human PCCA (DTC346), or a rAAV vector plasmid with a nucleotide sequence encoding the codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC 349).

Fig. 4A is a photograph showing protein expression levels detected by Western blotting in continuous liver cell lines (HepG2) transfected with a control plasmid (a plasmid with an empty vector, without TX), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA (DTC346), a rAAV vector plasmid with a nucleotide sequence encoding a codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC349), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA and a nucleotide sequence encoding the complete native sequence of human PCCB (DTC365), or a rAAV vector plasmid with a nucleotide sequence encoding enhanced green fluorescent protein eGFP (DTC343), respectively, from lane 1 to lane 7. Beta-actin ("actin") was used as a loading control.

Fig. 4B is a photograph showing the protein expression level detected by Western blotting in serial hepatocyte lines (Huh7) transfected with a control plasmid (a plasmid with an empty vector without TX), a rAAV vector plasmid with a nucleotide sequence encoding the enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA (DTC346), a rAAV vector plasmid with a nucleotide sequence encoding the codon-optimized sequence of human PCCA (DTC347, DTC348 or DTC349), or a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA and a nucleotide sequence encoding the complete native sequence of human PCCB (DTC365), respectively, from lane 1 to lane 7. Beta-actin ("actin") was used as a loading control.

FIG. 5 is a photograph showing the levels of PCCA protein expression detected by Western blotting in Whole Cell Lysates (WCL), subcellular fractions (cytosolic fraction (cyto) and mitochondrial fractions (mitochondria)) isolated from continuous liver cell lines (HepG2) transfected with or without a rAAV vector plasmid (DTC346) carrying a nucleotide sequence encoding the entire native sequence of human PCCA. 1X and 2X represent the relative volumes of the loaded mitochondrial fractions. Beta-actin ("actin") was used as a loading control.

Figure 6 is a bar graph showing, from left to right, the fold change in PCCA AAV9 titers (in Genomic Copies (GC)/mL) determined by qPCR after triple transfection of a continuous renal cell line (HEK293) transfected with a rAAV vector plasmid (DTC343) with nucleotide sequences encoding the enhanced green fluorescent protein eGFP, a rAAV vector plasmid (DTC346) with nucleotide sequences encoding the complete native sequence of human PCCA, or a rAAV vector plasmid (DTC347, DTC348, or DTC349) with nucleotide sequences encoding codon-optimized sequences of human PCCA, each plasmid being expressed in cells co-transfected with a plasmid expressing the AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral helper functions (pAdHelper). Untransfected cells were used as controls and were denoted "without TX".

Fig. 7 is a photograph of DNA alkaline sepharose from left to right of AAV9 particles of affinity-purified AAV (raav) vector cassettes containing a recombinant AAV (raav) vector cassette expressing PCCA and PCCB (DTC365), a recombinant AAV (raav) vector cassette expressing PCCA (DTC349, DTC348, DTC347 or DTC346), or a recombinant AAV (raav) vector cassette expressing eGFP (DTC343), respectively.

Figure 8 is a bar graph showing fold changes in PCCB RNA expression determined by RT-qPCR after transfection of continuous liver cell lines (HepG2) with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmid with nucleotide sequences encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmid with nucleotide sequences encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmid with nucleotide sequences encoding codon-optimized sequences of human PCCB (DTC367, DTC370, DTC368, DTC369, or DTC 371).

Fig. 9A is a photograph showing the level of protein expression detected by Western blotting in continuous liver cell lines (HepG2) transfected with a control plasmid (plasmid with empty vector, no TX), a rAAV vector plasmid with a nucleotide sequence encoding an enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the entire native sequence of human PCCB (DTC366), or a rAAV vector plasmid with a nucleotide sequence encoding a codon-optimized sequence of human PCCB (DTC367, DTC368, DTC369, DTC370, or DTC371), respectively, from lane 1 to lane 8. Beta-actin ("actin") was used as a loading control.

Fig. 9B is a photograph showing the level of protein expression detected by Western blotting in serial hepatocyte lines (Huh7) transfected with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmid with nucleotide sequence encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmid with nucleotide sequence encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmid with nucleotide sequence encoding codon optimized sequence of human PCCB (DTC367, DTC369, DTC368, DTC370, or DTC371), respectively, from lane 1 to lane 8. Beta-actin ("actin") was used as a loading control.

Figure 10 is a photograph showing the levels of PCCB protein expression detected by Western blot in Whole Cell Lysates (WCL), subcellular fractions (cytosolic fraction (cyto) and mitochondrial fractions (mitochondria)) isolated from continuous liver cell lines (HepG2) transfected with or without a rAAV vector plasmid (DTC366) carrying the nucleotide sequence encoding the entire native sequence of human PCCB. 1X and 2X represent the relative volumes of the loaded mitochondrial fractions. Beta-actin ("actin") was used as a loading control.

Figure 11 is a bar graph showing, from left to right, the fold change in PCCB AAV9 titers (in Genomic Copies (GC)/mL) determined by qPCR after triple transfection of a continuous renal cell line (HEK293) transfected with a rAAV vector plasmid (DTC343) with nucleotide sequences encoding the enhanced green fluorescent protein eGFP, a rAAV vector plasmid (DTC366) with nucleotide sequences encoding the complete native sequence of human PCCB, or a rAAV vector plasmid (DTC367, DTC368, DTC369, DTC370, or DTC371) with nucleotide sequences encoding codon-optimized sequences of human PCCA, each plasmid being expressed in cells co-transfected with a plasmid expressing AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral-assisted function (pAdHelper). Untransfected cells were used as controls and were denoted "without TX".

Fig. 12 is a photograph of DNA alkaline agarose gels from left to right of affinity purified AAV9 particles containing a control plasmid (plasmid with empty vector, no TX), a recombinant AAV (raav) vector cassette expressing eGFP (DTC343), or a recombinant AAV (raav) vector cassette expressing PCCB (DTC366, DTC367, DTC368, DTC369, DTC370, or DTC371), respectively.

Figure 13 is a bar graph showing the percentage of human PCCA protein expression relative to mouse endogenous PCCA expression in a wild type FVB mouse following rAAV treatment with either the complete native sequence encoding human PCCA (DTC346) or the codon optimized sequence of human PCCA (DTC347, DTC348 or DTC 349). Error bars indicate standard deviation.

Figure 14 is a bar graph showing the concentration (in nmol/g protein) of human wild-type PCCA protein expressed in a mouse model of sub-allele PCCA (a138T mutant) following rAAV treatment with a rAAV encoding the complete native sequence of human PCCA (DTC346) or a codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC 349). The percentages listed represent human wild-type PCCA expression calculated as a percentage of endogenous PCCA protein levels relative to wild-type FVB mice. Error bars indicate standard deviation.

Figure 15 is a bar graph showing human PCCA activity as measured by mean Count Per Minute (CPM) data in a mouse model of subthreshold allele PCCA (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Error bars indicate standard deviation. The percentages listed describe CPM data relative to that observed in wild-type FVB mice. Indicates p <0.01 compared to PBS treated group using Dunnett's multiple comparison test.

Figures 16A-C are bar graphs showing the concentration of known propionic acid blood biomarkers in a sub-allele PCCA mouse model (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Figure 16A is a bar graph showing plasma concentrations of C3 (propionyl carnitine) in a mouse model of sub-effective allele PCCA (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Figure 16B is a bar graph of plasma C3/C2 concentration ratios (propionyl-carnitine/acetyl-carnitine) in a mouse model of sub-allele PCCA (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Figure 16C is a bar graph showing plasma concentrations of 2-methyl citrate (2MC) in a sub-allele PCCA mouse model (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Pre represents 0 to 14 days before rAAV injection, 2W represents 2 weeks after rAAV injection, 3W represents 3 weeks after rAAV injection, 4W represents 4 weeks after rAAV injection, and 6W represents 6 weeks after rAAV injection. Error bars indicate standard deviation. P <0.01 in PBS treated group, p <0.001 in PBS treated group using Dunnett's multiple comparison test.

Detailed Description

The present invention provides a number of novel agents and compositions for therapeutic applications. As described herein, the nucleic acid sequences, vectors, recombinant viruses, and related compositions of the invention can be used to improve, prevent, or treat PA.

Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of the terms commonly used in molecular biology can be found in the following documents: benjamin Lewis, Gene V (Genes V), Oxford University Press publication, 1994(ISBN 0-19-854287-9); kendrew et al, Encyclopedia of Molecular Biology (The Encyclopedia of Molecular Biology), Blackwell Science Ltd, published 1994(ISBN 0-632-02182-9); robert A. Meyers, ed., "detailed Table-top Reference for Molecular Biology and Biotechnology" (Molecular Biology and Biotechnology: a Comprehensive Desk Reference), VCH Publishers, Inc. published 1995(ISBN 1-56081-.

To facilitate a review of the various embodiments of the present disclosure, the following explanation of specific terms is provided:

adeno-associated virus (AAV): a small replication-defective non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease, and it elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells, and can persist in an extrachromosomal state without integrating into the genome of the host cell. These properties make AAV an attractive viral vector for gene therapy. There are currently 12 recognized AAV serotypes (AAV 1-12).

Application: an agent, such as a therapeutic agent (e.g., a recombinant AAV), is provided or administered to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intratubular, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

Codon-optimized: "codon-optimized" nucleic acid refers to a nucleic acid sequence that has been altered to make codons most suitable for expression in a particular system (e.g., a particular species or group of species). For example, the nucleic acid sequence may be optimized for expression in mammalian cells or in a particular mammalian species (e.g., human cells). Codon optimization does not change the amino acid sequence of the encoded protein.

Enhancer: a nucleic acid sequence for increasing the transcription rate by increasing the activity of a promoter.

An intron: a piece of DNA that does not contain protein-encoding information within the gene. Introns are removed prior to translation of messenger RNA.

Inverted Terminal Repeat (ITR): the genome of adeno-associated virus is a symmetrical nucleic acid sequence required for efficient replication. The ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as origins of replication for viral DNA synthesis and are an essential cis component for the production of AAV integrating vectors.

Separating: an "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified away from other biological components in the cells or tissues of the organism in which the component naturally occurs or the organism itself, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells. Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins produced by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

The operable connection is as follows: a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in functional association with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.

A pharmaceutically acceptable carrier: pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington pharmaceuticals (Remington's Pharmaceutical Sciences), e.w. martin, Mack Publishing co., Easton, Pa., 15 th edition (1975) describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids as a medium, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions such as powder, pill, tablet or capsule forms, conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Prevention, treatment or amelioration of diseases: "preventing" a disease (e.g., PA) refers to inhibiting the overall progression of the disease. "treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition (e.g., PA) after it has begun to develop. By "improving" is meant reducing the number or severity of signs or symptoms of a disease (e.g., PA).

A promoter: a DNA region that directs/initiates transcription of a nucleic acid (e.g., a gene). Promoters include the necessary nucleic acid sequences adjacent to the transcription start site. Many promoter sequences are known to the person skilled in the art, and even combinations of different promoter sequences are possible in artificial nucleic acid molecules. As used herein, a gene-specific endogenous promoter refers to an endogenous promoter element that regulates the expression of an endogenous gene of interest. In an exemplary embodiment, the PCCA gene-specific endogenous promoter regulates expression of the PCCA gene. In another exemplary embodiment, the PCCB gene-specific endogenous promoter regulates expression of the PCCB gene.

Purification of: the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is completely or partially separated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to peptides, proteins, viruses, or other active compounds that have been separated from cells, cell culture media, or other crude preparations and fractionated to remove various components such as proteins, cell debris, and other components of the initial preparation.

Recombinant: a recombinant nucleic acid molecule is a nucleic acid molecule having a sequence that does not occur naturally or has a sequence that is made by the artificial combination of two otherwise separate sequence segments. Such artificial combination can be achieved by chemical synthesis or by artificial manipulation of the isolated nucleic acid molecule segments, for example by genetic engineering techniques.

Likewise, a recombinant virus is a virus that comprises sequences (e.g., genomic sequences) that are not naturally occurring or that have been made by the artificial combination of at least two sequences of different origin. The term "recombinant" also includes nucleic acids, proteins and viruses that are altered solely by the addition, substitution or deletion of a portion of a native nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule, e.g., a recombinant nucleic acid molecule encoding PCCA and/or a recombinant nucleic acid molecule encoding PCCB, has been packaged.

Sequence identity: the identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more identical the sequence. Sequence similarity can be measured in terms of percent similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more pronounced when the orthologous protein or cDNA is derived from a more closely related species (e.g., human and mouse sequences) than from a more closely related species (e.g., human and caenorhabditis elegans sequences).

Methods of sequence alignment for comparison purposes are well known in the art. Various programs and alignment algorithms are described in the following documents: smith & Waterman, adv.Appl.Math.2:482,1981; needleman & Wunsch, J.mol.biol.48:443,1970; pearson & Lipman, proc.natl.acad.sci.usa 85:2444,1988; higgins & Sharp, Gene,73: 237-; higgins & Sharp, CABIOS5:151-3, 1989; corpet et al, Nuc. acids Res.16:10881-90, 1988; huang et al, Computer applications. in the Biosciences 8,155-65, 1992; and Pearson et al, meth.mol.Rio.24:307-31, 1994. Detailed considerations for sequence alignment methods and homology calculations are presented by Altschul et al, J.mol.biol.215: 403-.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.mol.biol.215: 403-. Other information may be found on the NCBI website.

Serotype: a group of closely related microorganisms (e.g., viruses) that are distinguished by a set of characteristic antigens.

Stuffer sequence: refers to a nucleotide sequence contained within a larger nucleic acid molecule (e.g., a vector) that is typically used to create a desired separation or extend the nucleic acid molecule between two nucleic acid components (e.g., between a promoter and a coding sequence) so that it has a desired length. Stuffer sequences contain no protein coding information and may be of unknown/synthetic origin and/or unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

Subject: living multicellular vertebrate organisms, a category that includes both human and non-human mammals.

The synthesis comprises the following steps: produced by artificial means in the laboratory, for example, synthetic nucleic acids can be chemically synthesized in the laboratory.

Untranslated region (UTR): typical mRNAs contain a5 'untranslated region ("5' UTR") and a 3 'untranslated region (3' UTR) upstream and downstream of the coding region, respectively (see Mignone F. et al, (2002) Genome Biol 3: REVIEWS 0004).

A therapeutically effective amount of: an amount of a given drug or therapeutic agent (e.g., a recombinant AAV) sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of an agent depends on several factors, including but not limited to the subject or cell being treated and the mode of administration of the therapeutic composition.

Carrier: a vector is a nucleic acid molecule that allows insertion of a foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. The vector may include nucleic acid sequences, such as an origin of replication, which allow it to replicate in the host cell. The vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences that allow transcription and translation of the inserted gene or genes. In certain embodiments herein, the vector is an AAV vector.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. No specific number of an indication includes a plural indication unless the context clearly dictates otherwise. "comprising A or B" is meant to include A or B or both A and B. It is also understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values provided for a nucleic acid or polypeptide are approximate and provided for descriptive purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Viral vector (b):

in certain instances, the present disclosure provides adeno-associated virus (AAV) vectors comprising a packaged genome comprising AAV5 '-ITRs, promoter sequences, partial or complete coding sequences of PCCA or its isoforms or functional fragments or functional variants thereof, and AAV 3' -ITRs.

In certain embodiments, the disclosure provides an AAV vector comprising a packaged genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of the human PCCA 5' -UTR, a partial or complete coding sequence of PCCA or its isoform or a functional fragment or functional variant thereof, a truncated or complete nucleotide sequence of the human PCCA 3 '-UTR, and an AAV 3' -ITR.

In certain instances, the disclosure provides AAV vectors comprising a packaged genome comprising AAV5 '-ITRs, promoter sequences, partial or complete coding sequences of PCCB or its isoforms or functional fragments or functional variants thereof, and AAV 3' -ITRs.

In certain embodiments, the disclosure provides an AAV vector comprising a packaged genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of human PCCB 5' -UTR, a partial or complete coding sequence of PCCB or an isoform or functional fragment or functional variant thereof, a truncated or complete nucleotide sequence of human PCCB 3 '-UTR, and an AAV 3' -ITR.

In certain embodiments, the packaged genome may further comprise an enhancer, intron, consensus Kozak sequence, and/or polyadenylation signal as described herein. In certain embodiments, the recombinant vector may further comprise one or more stuffer nucleic acid sequences. In one embodiment, the stuffer nucleic acid sequence is located between the intron and a partial or complete coding sequence of PCCA or PCCB.

In various embodiments described herein, the recombinant viral vector is an AAV vector. The AAV vector can be any of AAV vectors of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12), as well as any of more than 100 variants isolated from human and non-human primate tissue. See, e.g., Choi et al, 2005, Curr Gene ther.5: 299-. Any serotype of AAV vector may be used in the present invention, and the selection of AAV serotype depends in part on the cell type targeted by the gene therapy. For the treatment of PA, the liver is one of the relevant target organs. In certain embodiments, the AAV vector is selected from serotype 9(AAV9), serotype 8(AAV8), or a variant thereof. In exemplary embodiments, the AAV vector is serotype 9(AAV9) or a variant thereof.

In certain embodiments, the recombinant AAV vector comprises AAV ITR sequences that serve both as a vector DNA origin of replication and as a packaging signal for the vector genome when AAV and adenoviral helper functions are provided in trans. In addition, the ITRs serve as targets for single-strand endonucleolytic nicks of large Rep proteins, resolving individual genomes from replicative intermediates.

In certain embodiments, the 5' -ITR sequence is from AAV 2. In certain embodiments, the 3' -ITR sequence is from AAV 2. In certain embodiments, the 5 '-ITR and 3' -ITR sequences are from AAV 2. In certain embodiments, the 5 '-ITR sequence and/or the 3' -ITR sequence is from AAV2 and comprises SEQ ID NO: 15 or consist thereof. In other embodiments, the 5 '-ITR and/or 3' -ITR sequences are from a non-AAV 2 source.

In certain exemplary embodiments, the AAV vector is an AAV serotype 9(AAV9) vector, and the vector comprises an enhancer, promoter, intron, coding sequence for PCCA or PCCB, and a polyadenylation signal as described herein. In certain embodiments, the AAV9 vector further comprises two AAV2, AAV8, or AAV9 Inverted Terminal Repeat (ITR) sequences: one 5 'to the enhancer and one 3' to the polyadenylation signal. In exemplary embodiments, the AAV9 vector comprises two AAV2 Inverted Terminal Repeat (ITR) sequences: one 5 'to the enhancer and one 3' to the polyadenylation signal. In certain embodiments, the AAV2 ITR sequence comprises SEQ ID NO: 15 or consist thereof. In another exemplary embodiment, the AAV9 vector comprises two AAV9 Inverted Terminal Repeat (ITR) sequences: one 5 'to the enhancer and one 3' to the polyadenylation signal.

A promoter:

in various aspects described herein, viral vectors are provided that contain a packaged genome comprising a promoter sequence that helps drive and regulate expression of a transgene, such as PCCA or PCCB. In certain embodiments, the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a Cytomegalovirus (CMV) immediate early gene promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, a CAG promoter, a PCCA gene-specific endogenous promoter, and a PCCB gene-specific endogenous promoter. In exemplary embodiments, the promoter sequence is located between the selected 5' -ITR sequence and part or the entire coding sequence of PCCA or PCCB. In certain embodiments, the promoter sequence is located downstream of the enhancer sequence. In certain embodiments, the promoter sequence is located upstream of the intron sequence. In certain embodiments, the promoter sequence is located between the selected 5 '-ITR sequence and a truncated or complete nucleotide sequence of the human PCCA or human PCCB 5' -untranslated region (UTR).

In certain illustrative embodiments, a ubiquitous chicken β -actin promoter (CBA) is used, which may optionally be located downstream of the CMV immediate early enhancer (CMV IE). In certain embodiments, a packaged genome described herein comprises an indigenous PCCA or PCCB promoter element. In certain illustrative embodiments, when part or all of the coding sequence in the packaged genome is for PCCA, the packaged genome described herein comprises a PCCA gene-specific endogenous promoter comprising a nucleotide sequence that is identical to the nucleotide sequence of SEQ ID NO: 34, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length. In certain embodiments, the packaged genome described herein comprises a PCCA gene-specific endogenous promoter comprising a nucleotide sequence that is identical to SEQ ID NO: 34 (e.g., about 30, about 45, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, or about 750). In certain illustrative embodiments, when part or all of the coding sequence in the vector genome is for PCCA, the packaged genome described herein comprises a PCCA gene-specific endogenous promoter comprising a nucleotide sequence that is identical to the nucleotide sequence of SEQ ID NO: 34, and a nucleotide sequence of at least 15 contiguous nucleotides with 100% identity over a region of equal length.

In certain illustrative embodiments, when part or all of the coding sequence in the vector genome is for PCCB, the packaged genome described herein comprises a PCCB gene-specific endogenous promoter comprising a sequence identical to the sequence of SEQ ID NO: 36, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length. In certain embodiments, the packaged genome described herein comprises a PCCB gene-specific endogenous promoter comprising a promoter that is identical to SEQ ID NO: 36 (e.g., about 30, about 45, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, or about 450) of at least 95% identity over an equal length region. In certain illustrative embodiments, when part or all of the coding sequence in the vector genome is for PCCB, the packaged genome described herein comprises a PCCB gene-specific endogenous promoter comprising a sequence identical to the sequence of SEQ ID NO: 36, and a nucleotide sequence of at least 15 contiguous nucleotides with 100% identity over a region of equal length.

In certain embodiments, the promoter is selected from the group consisting of chicken β -actin (CBA) promoter, Cytomegalovirus (CMV) immediate early gene promoter, transthyretin (TTR) promoter, thyroxine-binding globulin (TBG) promoter, α -1 antitrypsin factor (A1AT) promoter, and CAG promoter. In an exemplary embodiment, the promoter is a CBA promoter. In one embodiment, the CBA promoter comprises SEQ ID NO: 18 or consist thereof.

In addition to promoters, the packaged genome may contain other suitable transcription initiation, termination, enhancer sequences, and high efficiency RNA processing signals. As described in further detail below, such sequences include splicing and polyadenylation (poly a) signals, regulatory elements to enhance expression (i.e., WPRE), sequences to stabilize cytoplasmic mRNA, sequences to enhance translation efficiency (i.e., Kozak consensus sequence), and sequences to enhance protein stability.

In certain embodiments, the packaged genome further comprises a consensus Kozak sequence. In certain embodiments, the consensus Kozak sequence is located downstream of an intron sequence. In one embodiment, the consensus Kozak sequence is GCCGCC (SEQ ID NO: 24). As will be appreciated by those skilled in the art, the consensus Kozak sequence is typically immediately upstream of the coding sequence; in this case immediately upstream of the partial or complete coding sequence of PCCA or PCCB. As will be appreciated by those skilled in the art, the consensus Kozak sequence may be considered to share ATG residues corresponding to the start codon of a therapeutic polypeptide, such as PCCA or PCCB. For simplicity of disclosure, the consensus Kozak sequence described herein comprises a sequence of 6 nucleotides corresponding to a region not shared with the therapeutic polypeptide, e.g., PCCA or PCCB.

Untranslated region (UTR):

the 5' -untranslated region (UTR) from endogenous gene-specific mRNAs is known to play an important role in optimizing transgene production by competing with intracellular transcripts for translation initiation factors and ribosomes, increasing mRNA half-life by minimizing mRNA attenuation or post-transcriptional gene silencing, and avoiding deleterious interactions with regulatory proteins or inhibitory RNA secondary structures (see Chiba, Y. and Green, P. (2009) J. plant biol.52, 114-124; Moore, M.J. and Proudfoot, N.J. (2009) Cell 136, 688-700; Jackson, R.J. et al, (2010) Nat. Rev. mol. Cell biol.11, 113-127). The 3 '-untranslated region (3' -UTR) located downstream of the protein coding sequence has been found to be involved in a number of regulatory processes, including transcript cleavage, stability and polyadenylation, translation and mRNA localization. They are therefore crucial in determining the fate of mRNA (see Barrett, L.W. et al, (2012) Cell Mol Life Sci. Nov; 69(21): 3613-. In certain embodiments, the disclosure provides a rAAV comprising a packaged genome comprising a truncated or complete nucleotide sequence of a human PCCA or PCCB 5' -UTR. In certain embodiments, the disclosure provides a rAAV comprising a packaged genome comprising a truncated or complete nucleotide sequence of a human PCCA or PCCB 3' -UTR.

In certain embodiments, the disclosure provides a rAAV comprising a packaged genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of a human PCCA 5' -UTR, a partial or complete coding sequence of PCCA or an isoform or functional fragment or functional variant thereof, a truncated or complete human PCCA 3 '-UTR nucleotide sequence, and an AAV 3' -Inverted Terminal Repeat (ITR).

In certain embodiments, the disclosure provides a rAAV comprising a packaged genome comprising an AAV5 '-ITR, a promoter sequence, a truncated or complete nucleotide sequence of a human PCCB 5' -UTR, a partial or complete coding sequence of a PCCB or isoform or functional fragment or functional variant thereof, a truncated or complete human PCCB 3 '-UTR nucleotide sequence, and an AAV 3' -ITR.

In certain embodiments, the packaged genome comprises a nucleotide sequence consisting of SEQ ID NO: 30, intact human PCCA 5' -UTR. In certain embodiments, the packaged genome comprises a nucleotide sequence consisting of SEQ ID NO: 32, complete human PCCB 5' -UTR.

In certain embodiments, the packaged genome comprises a truncated native human PCCA 5' -UTR. In certain embodiments, the truncated human PCCA 5' -UTR comprises a sequence identical to SEQ ID NO: 30 to about 1400 consecutive nucleotides (e.g., about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 275, about 300, about 325, about 350, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 900, about 925, about 950, about 975, about 1000, about 1025, about 1050, about 1075, about 1100, about 1125, about 1150, about 1175, about 1200, about 1225, about 1250, about 1275, about 1300, about 1325, or about 1350).

In certain embodiments, the packaged genome comprises a truncated native human PCCB 5' -UTR. In certain embodiments, the truncated human PCCB 5' -UTR comprises a sequence identical to SEQ ID NO: 32 from about 50 contiguous nucleotides of at least 95% identity to about 1400 contiguous nucleotides (e.g., about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 275, about 300, about 325, about 350, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 900, about 925, about 950, about 975, about 1000, about 1025, about 1050, about 1075, about 1100, about 1125, about 1150, about 1175, about 1200, about 1225, about 1250, about 1275, about 1300, about 1325, or about 1350).

In certain embodiments, the packaged genome comprises a nucleotide sequence consisting of SEQ ID NO: 31, the complete human PCCA 3' -UTR. In certain embodiments, the packaged genome comprises a nucleotide sequence consisting of SEQ ID NO: 33, complete human PCCB 3' -UTR.

In certain embodiments, the packaged genome comprises a truncated native human PCCA 3' -UTR. In certain embodiments, the truncated human PCCA 3' -UTR comprises a sequence identical to SEQ ID NO: 31 of about 15 contiguous nucleotides (e.g., about 30, about 45, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270) that are at least 95% identical.

In certain embodiments, the packaged genome comprises a truncated native human PCCB 3' -UTR. In certain embodiments, the truncated human PCCB 3' -UTR comprises a sequence identical to SEQ ID NO: 33, about 15 contiguous nucleotides (e.g., about 30, about 45, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150) that are at least 95% identical over a region of equal length.

PCCA or PCCB polypeptides:

as described herein, various aspects of the invention provide a recombinant vector comprising a packaged genome comprising AAV5 '-ITRs, promoter sequences, partial or complete coding sequences of PCCA or isoforms or functional fragments or functional variants thereof, and AAV 3' -ITRs. Also described herein are recombinant vectors containing a packaged genome comprising AAV5 '-ITRs, a promoter sequence, a partial or complete coding sequence for PCCB or an isoform or a functional fragment or functional variant thereof and AAV 3' -ITRs.

In one embodiment, the partial or complete coding sequence of PCCA or PCCB is a wild-type coding sequence. As used herein, the term "wild-type" refers to a biopolymer (e.g., a polypeptide sequence or a polynucleotide sequence) that is identical to a biopolymer (e.g., a polypeptide sequence or a polynucleotide sequence) that occurs in nature.

In alternative embodiments, the partial or complete coding sequence of PCCA or PCCB is a codon optimized coding sequence. In one embodiment, the partial or complete coding sequence of PCCA or PCCB is codon optimized for expression in humans.

In various embodiments described herein, a vector is provided comprising a packaged genome comprising a coding sequence for PCCA or PCCB. Polypeptides delivered using the vectors described herein encompass PCCA and PCCB polypeptides that may be useful in the treatment of mammals, including humans.

In certain embodiments, the polypeptide expressed using the vectors described herein is PCCA (SEQ ID NO: 16; GenBank accession number NP-000273.2; 728 amino acids), or a functional fragment, functional variant, or functional isoform thereof. In certain embodiments, the polypeptide expressed with the vector described herein is PCCA and comprises SEQ ID NO: 16 or consist thereof.

In one embodiment, the PCCA polypeptide consists of SEQ ID NO: 1, or a wild-type coding sequence shown in seq id no. In another embodiment, coding sequences expressing native isoforms or variants of PCCA may be used, such as shown in UniProtKB/Swiss-Prot accession numbers P05165-1(SEQ ID NO: 25), P05165-2(SEQ ID NO: 26), and P05165-3(SEQ ID NO: 27). In alternative embodiments, the PCCA polypeptide is encoded by a codon-optimized coding sequence. In certain embodiments, the PCCA polypeptide consists of a sequence identical to SEQ ID NO: 1, the coding sequence of codon optimization having less than 80% identity to the wild-type coding sequence shown in 1. In certain exemplary embodiments, the PCCA polypeptide consists of a sequence selected from SEQ ID NOs: 2-6. In certain embodiments, the coding sequence for PCCA may further comprise a stop codon (TGA, TAA or TAG) at the 3' terminus.

In certain embodiments, the polypeptide expressed using the vectors described herein is PCCB (SEQ ID NO: 17; GenBank accession number NP-000523.2; 539 amino acids) or a functional fragment, functional variant or functional isoform thereof. In certain embodiments, the polypeptide expressed with the vector described herein is PCCB and comprises SEQ ID NO: 17 or consist thereof.

In one embodiment, the PCCB polypeptide consists of SEQ ID NO: 7, or a wild-type coding sequence shown in seq id no. In another embodiment, coding sequences expressing native isoforms or variants of PCCB may be used, such as shown in UniProtKB/Swiss-Prot accession numbers P05166-1(SEQ ID NO: 28) and P05166-2(SEQ ID NO: 29). In alternative embodiments, the PCCB polypeptide is encoded by a codon optimized coding sequence. In certain embodiments, the PCCB polypeptide consists of a sequence identical to SEQ ID NO: 7, which is less than 80% identical to the wild-type coding sequence. In certain exemplary embodiments, the PCCB polypeptide consists of a sequence selected from SEQ ID NOs: 8-12. In certain embodiments, the coding sequence of the PCCB may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus.

In various instances, the invention can be used to deliver fragments, variants, isoforms, or fusions of PCCA or PCCB polypeptides described herein.

In certain embodiments, the invention can be used to deliver a PCCA or PCCB polypeptide fragment comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acid residues and retaining one or more activities associated with the full-length polypeptide (e.g., catalytic activity in the case of an enzyme). Such fragments may be obtained by recombinant techniques which are conventional and well known in the art. Furthermore, such fragments can be tested by conventional in vitro assays known to the skilled artisan. For example, propionyl-coa carboxylase (PCC) activity can be determined by the following steps: (1) diluting said polypeptide in 10mM phosphate buffer (pH 7.0) containing 1mM 2-mercaptoethanol and 0.1mg/ml bovine serum albumin, (2) removing the buffer containing 50mM Tris-HCl pH8.0, 5mM glutathione, 2mM ATP、100mM KCl、10mM MgCl₂、10mM[¹⁴C]Bicarbonate (specific activity 12.4mCi/mmol), 3mM propionyl-coa, standard reaction mixture and incubation with enzyme at 37 ℃ for 15min, (3) stop the reaction by addition of 10% trichloroacetic acid, (4) centrifuge at 200x g, (5) dry aliquot under heat lamp, (6) dissolve in water, and (7) count in Aquasol, where one unit of enzyme activity is defined as the amount of enzyme catalyzing 1pmol bicarbonate fixation per minute at 37 ℃. For a description of the PCC activity assay, see Kaluusek et al, 1980, J Biol Chem255(1):60-65 and Hsia et al, 1973J. Peditar. 83: 625-. The invention also includes nucleic acid molecules encoding the polypeptide fragments described above.

In certain embodiments, the invention can be used to deliver variants of the PCCA or PCCB polypeptides. In certain embodiments, the variant polypeptide may be identical to a wild-type therapeutic polypeptide, such as SEQ ID NO: 16 or a wild-type PCCA polypeptide of SEQ ID NO: 17 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) is at least 80% identical. In certain embodiments, the variant therapeutic polypeptide may have at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 different residues as compared to the corresponding wild-type polypeptide. Such variants may be obtained by recombinant techniques which are conventional and well known in the art. Furthermore, such variants can be tested for catalytic activity by conventional in vitro assays known to the skilled artisan. For a description of propionyl-CoA carboxylase activity assays see, e.g., Kaluusek et al, 1980, J Biol Chem255(1):60-65 and Hsia et al, 1973J. Peditar. 83: 625) 628. The invention also includes nucleic acid molecules encoding the above therapeutic polypeptide variants.

Novel codon optimized sequences:

in certain instances, the present disclosure provides novel codon-optimized nucleic acid sequences encoding PCCA. In one embodiment, the PCCA-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NO: 1 by less than 80%. In certain embodiments, the PCCA-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NOs: 2-6 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) identity. In certain embodiments, the codon-optimized nucleic acid sequence encoding PCCA is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-6, 100% identity. In certain embodiments, the present disclosure provides a polypeptide that differs from SEQ ID NO: 1, and less than 80% identical to the wild-type coding sequence shown in SEQ ID NOs: 2-6 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In exemplary embodiments, the present disclosure provides a polypeptide selected from SEQ ID NOs: 2-6 encoding PCCA. Also provided are SEQ ID NOs: 2-6, which encodes a polypeptide having functional PCCA activity. In certain embodiments, the nucleic acid sequence encoding PCCA may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus.

In certain instances, the present disclosure provides novel codon-optimized nucleic acid sequences encoding PCCBs. In one embodiment, the PCCB-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NO: 7 is less than 80% identical. In certain embodiments, the PCCB-encoding codon-optimized nucleic acid sequence is identical to SEQ ID NOs: 8-12 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) identity. In certain embodiments, the PCCB-encoding codon-optimized nucleic acid sequence is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: sequence 100% identity from 8 to 12. In certain embodiments, the present disclosure provides a polypeptide that differs from SEQ ID NO: 7, and less than 80% identical to the wild-type coding sequence shown in SEQ ID NOs: 8-12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In exemplary embodiments, the present disclosure provides a polypeptide selected from SEQ ID NOs: 8-12 encoding a PCCB. Also provided are the nucleic acids set forth in SEQ ID NOs: 8-12 encoding a polypeptide having functional PCCB activity. In certain embodiments, the nucleic acid sequence encoding PCCB may further comprise a stop codon (TGA, TAA, or TAG) at the 3' terminus.

Vector genomic elements:

in certain embodiments, the rAAV contains a packaged vector genome, which further comprises one or more enhancer sequences. In one embodiment, the enhancer is selected from the cytomegalovirus immediate early gene (CMV) enhancer, the transthyretin enhancer (enTTR), the chicken β -actin (CBA) enhancer, the En34 enhancer, and the ApoE enhancer. In an exemplary embodiment, the enhancer is a CMV enhancer. In one embodiment, the CMV enhancer comprises SEQ ID NO: 19 or consist thereof.

In certain embodiments, the rAAV contains a packaged vector genome, which further comprises one or more intron sequences. In one embodiment, the intron is selected from the group consisting of the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, and the hFIX intron. In an exemplary embodiment, the intron is the SV40 small T intron. In one embodiment, the SV40 small T intron sequence comprises SEQ ID NO: 20 or consist thereof. In another exemplary embodiment, the intron is a rHBB intron. In one embodiment, the rHBB intron sequence comprises SEQ ID NO: 21 or consist thereof.

In certain embodiments, the rAAV contains a packaged vector genome, which further comprises a polyadenylation signal sequence. In one embodiment, the polyadenylation signal sequence is selected from the group consisting of a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, a rabbit β globin polyadenylation signal sequence, a PCCA gene-specific endogenous polyadenylation signal sequence, a PCCB gene-specific endogenous polyadenylation signal sequence. In an exemplary embodiment, the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence. In one embodiment, the BGH polyadenylation signal sequence comprises SEQ ID NO: 22 or consist thereof. In another exemplary embodiment, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence. In one embodiment, the SV40 polyadenylation signal sequence comprises SEQ ID NO: 23 or consist thereof. In one embodiment, the polyadenylation signal sequence is a gene-specific endogenous polyadenylation sequence. In an exemplary embodiment, when part or all of the coding sequence in the vector genome is for PCCA, the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the PCCA gene-specific endogenous polyadenylation signal sequence comprises a sequence identical to SEQ ID NO: 35, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length. In certain embodiments, the PCCA gene-specific endogenous polyadenylation signal sequence comprises a sequence identical to SEQ ID NO: 35, about 15 contiguous nucleotides (e.g., about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or about 160) that are at least 95% identical over a region of equal length. In another exemplary embodiment, when part or all of the coding sequence in the vector genome is for PCCB, the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the PCCB gene-specific endogenous polyadenylation signal sequence comprises a nucleotide sequence identical to SEQ ID NO: 37, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length. In certain embodiments, the PCCA gene-specific endogenous polyadenylation signal sequence comprises a sequence identical to SEQ ID NO: 37, a nucleotide sequence of about 15 contiguous nucleotides (e.g., about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100) that are at least 95% identical over an equal length region.

AAV capsids:

in another aspect, the disclosure provides a rAAV useful as a gene therapy agent in the treatment of PA, wherein the rAAV comprises an AAV capsid and a vector genome described herein packaged therein. In certain embodiments, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10 or hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAVrh10, or AAVhu 37). In exemplary embodiments, the AAV vector is an AAV serotype 9(AAV9) vector, an AAV9 variant vector, an AAV serotype 8(AAV8) vector, or an AAV serotype 2(AAV2) vector.

The AAV9 capsid is a self-assembled AAV capsid composed of multiple AAV9 vp proteins. The AAV9 vp protein is typically expressed as a polypeptide represented by SEQ ID NO: 13 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity thereto, which nucleic acid sequence encodes an alternative splice variant of the nucleic acid sequence of SEQ ID NO: 14 (GenBank accession No. AAS 99264). These splice variants produce SEQ ID NO: 14, or a protein of different length. In certain embodiments, the AAV9 capsid comprises a nucleic acid sequence having identity to AAS 9926499 or to SEQ ID NO: 1499% identity to an amino acid sequence. See also U.S. Pat. No. 7,906,111 and WO/2005/033321. As used herein, AAV9 variants are included as described in, for example, WO/2016/049230, U.S. patent No. 8,927,514, U.S. patent publication No. 2015/0344911, and U.S. patent No. 8,734,809.

As indicated herein, the AAV9 sequences and proteins can be used to produce rAAV. However, in other embodiments, another AAV capsid is selected. Tissue specificity is determined by the capsid type. AAV serotypes transducing a suitable target (e.g., liver, muscle, lung, or CNS) can be selected as a source of capsid for AAV viral vectors, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64Rl, AAVrh64R2, AAVrh 8. See, e.g., U.S. patent publication No. 2007/0036760, U.S. patent publication No. 2009/0197338, and EP 1310571. See also WO 2003/042397(AAV7 and other simian AAV), us patent nos. 7282199 and 7790449(AAV 8). In addition, AAV that has not been discovered or recombinant AAV based thereon can be used as a source of AAV capsids. These documents also describe other AAVs that may be selected for production of the AAV, and are incorporated herein by reference. In certain embodiments, the AAV capsid used in the viral vector may be produced by mutation (i.e., by insertion, deletion, or substitution) of one of the AAV capsids described above or a nucleic acid encoding therefor. In certain embodiments, the AAV capsid is chimeric, comprising domains from two or three or four or more of the AAV capsid proteins described above. In certain embodiments, the AAV capsid is a chimera of Vpl, Vp2, and Vp3 monomers from two or three different AAV or recombinant AAV. In certain embodiments, the rAAV composition comprises more than one capsid as described above.

A host cell comprising a recombinant nucleic acid molecule:

in certain instances, provided herein are host cells comprising a recombinant nucleic acid molecule, viral vector, e.g., an AAV vector, or rAAV disclosed herein. In particular embodiments, the host cell may be suitable for propagation of AAV.

A wide range of host cells can be used, such as bacterial, yeast, insect, mammalian cells, and the like. In certain embodiments, the host cell may be a cell (or cell line) suitable for production of recombinant AAV (rAAV), such as HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19, or MRC-5 cells.

The recombinant nucleic acid molecule or vector may be delivered to the host cell culture using any suitable method known in the art. In certain embodiments, a stable host cell line is produced having the recombinant nucleic acid molecule or vector inserted into its genome. In certain embodiments, stable host cell lines containing the rAAV vectors described herein are produced. After transfection of the rAAV vector into a host culture, integration of the rAAV in the host genome can be determined by a variety of different methods, such as antibiotic selection, fluorescence activated cell sorting, southern blotting, PCR-based detection, fluorescence in situ hybridization, as described in: nakai et al, Nature Genetics (2003)34, 297-302; philpott et al, Journal of Virology (2002)76(11): 5411-5421; and Howden et al, J Gene Med 2008; 10:42-50. In addition, stable cell lines can be established according to procedures well known in the art, for example, in Clark, Kidney International Vol 61(2002): S9-S15 and Yuan et al, Human Gene Therapy 2011 May; 22(5) 613-24.

Recombinant AAV for use in gene therapy:

AAV belongs to the parvoviridae and dependovirus genera. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into the AAV capsid at the same frequency.

The AAV genome is characterized by two Inverted Terminal Repeats (ITRs) flanking two open reading frames (ORBs). For example, in the AAV2 genome, the first 125 nucleotides of an ITR are palindromes, which fold upon themselves to maximize base pairing and form a T-hairpin structure. The other 20 bases of the ITRs, referred to as D sequences, remain unpaired. ITRs are cis-acting sequences important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for the synthesis of the second strand by the DNA polymerase. The double stranded DNA formed during this synthesis, called replicative monomers, is used for a second round of self-initiated replication and forms replicative dimers. These double-stranded intermediates are processed by a strand displacement mechanism to produce single-stranded DNA for packaging and double-stranded DNA for transcription. Located within the ITRs are Rep binding elements and terminal dissociation sites (TRSs). These parts are used by the viral regulatory protein Rep to process the double stranded intermediates during AAV replication. In addition to their role in AAV replication, ITRs are essential for AAV genomic packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Days and Berns, Clin Microbiol Rev 21(4): 583-.

The left ORF of AAV contains the Rep gene, which encodes four proteins — Rep78, Rep68, Rep52, and Rep 40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP 3). The AAV capsid contains 60 viral capsid proteins arranged in icosahedral symmetry. VP1, VP2 and VP3 were present in a molar ratio of 1:1:10 (Daya and Berns, Clin Microbiol Rev 21(4): 583-.

AAV is currently one of the most commonly used viruses for gene therapy. Although AAV infects humans and certain other primate species, it is not known to cause disease, and it elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and persist extrachromosomally without integrating into the genome of the host cell. Because of the advantageous characteristics of AAV, the present disclosure contemplates the use of AAV in the recombinant nucleic acid molecules and methods disclosed herein.

AAV has several characteristics required for gene therapy vectors, including the ability to bind to and enter target cells, enter the nucleus, express for long periods in the nucleus, and low toxicity. However, the small size of the AAV genome limits the size of heterologous DNA that can be incorporated. To minimize this problem, AAV vectors have been constructed that do not encode Rep and Integration Efficiency Elements (IEEs). ITRs are retained because they are cis-signals required for packaging (Daya and Berns, Clin Microbiol Rev,21(4): 583-.

Methods for producing rAAV suitable for Gene therapy are well known in the art (see, e.g., U.S. patent application Nos. 2012/0100606, 2012/0135515, 2011/0229971, and 2013/0072548; and Ghosh et al, Gene Ther 13(4):321-329,2006), and are useful in the recombinant nucleic acid molecules and methods disclosed herein.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating Propionemia (PA), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In certain embodiments, the rAAV contains a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, a promoter sequence, a partial or complete coding sequence of PCCA or its isoform or a functional fragment or functional variant thereof and a 3' -ITR. In certain embodiments, the rAAV contains a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, promoter sequence, truncated or complete nucleotide sequence of human PCCA 5' -UTR, partial or complete coding sequence of PCCA or its isoform or functional fragment or functional variant thereof, truncated or complete nucleotide sequence of human PCCA 3 '-UTR and 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3' -ITR. Thus, in another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, enhancer sequence, promoter sequence, intron sequence, PCCA or its isoform or a functional fragment or functional variant thereof, polyadenylation signal sequence and 3' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, enhancer sequence, promoter sequence, intron sequence, truncated or complete nucleotide sequence of the human PCCA 5' -UTR, part or complete coding sequence of PCCA or its isoform or a functional fragment or functional variant thereof, polyadenylation signal sequence, truncated or complete nucleotide sequence of the human PCCA 3 '-UTR and 3' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: AAV 25 '-ITR sequence, CMV enhancer, CBA promoter, SV40 small T intron, PCCA coding sequence, BGH polyadenylation signal sequence and further AAV 23' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: AAV 25 '-ITR sequence, CMV enhancer, native PCCA promoter, SV40 small T intron, nucleotide sequence of PCCA 5' -UTR, coding sequence of PCCA, native PCCA polyadenylation signal sequence, nucleotide sequence of PCCA 3 '-UTR and AAV 23' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the native PCCA 5' -UTR sequence. In certain embodiments, the coding sequence for PCCA is selected from SEQ ID NO: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

The schematic provided in fig. 1 illustrates an exemplary packaged vector genomic construct for expressing PCCA, which shows, in 5 'to 3' order: 5 '-ITR, CMV enhancer, CBA promoter, SV40 small T intron, consensus Kozak sequence, PCCA coding sequence, SV40 polyadenylation signal sequence and 3' -ITR.

In certain instances, the present disclosure provides for the use of a rAAV disclosed herein for treating Propionemia (PA), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, wherein the packaged genome comprises as operably linked components, in 5 'to 3' order: 5 '-ITR, promoter sequence, PCCB or its isotype or its functional fragment or functional variant of partial or complete coding sequence and 3' -ITR. In certain embodiments, the rAAV contains a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, promoter sequence, truncated or complete nucleotide sequence of human PCCB 5' -UTR, partial or complete coding sequence of PCCB or its isoform or functional fragment or functional variant thereof, truncated or complete nucleotide sequence of human PCCB 3 '-UTR and 3' -ITR. In exemplary embodiments, the packaged genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3' -ITR. Thus, in another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, enhancer sequence, promoter sequence, intron sequence, PCCB or part or the complete coding sequence of its isoform or its functional fragment or functional variant, polyadenylation signal sequence and 3' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: 5 '-ITR, enhancer sequence, promoter sequence, intron sequence, truncated or complete nucleotide sequence of human PCCB 5' -UTR, partial or complete coding sequence of PCCB or its isoform or functional fragment or functional variant thereof, polyadenylation signal sequence, truncated or complete nucleotide sequence of human PCCB 3 '-UTR and 3' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: AAV 25 '-ITR sequences, CMV enhancer, CBA promoter, SV40 small T intron, coding sequence for PCCB, BGH polyadenylation signal sequence and AAV 23' -ITR. In another exemplary embodiment, the rAAV comprises a packaged genome comprising, in 5 'to 3' order as operably linked components: AAV 25 '-ITR sequence, CMV enhancer, native PCCB promoter, SV40 small T intron, nucleotide sequence of PCCB 5' -UTR, coding sequence of PCCB, native PCCB polyadenylation signal sequence, nucleotide sequence of PCCB 3 '-UTR and AAV 23' -ITR. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In certain embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the native PCCB 5' -UTR sequence. In certain embodiments, the coding sequence of the PCCB is selected from the group consisting of SEQ ID NOs: 7-12. In certain embodiments, the capsid is an AAV9 capsid.

The schematic provided in fig. 2 illustrates an exemplary packaged vector genomic construct for expressing PCCB, which shows, in 5 'to 3' order: 5 '-ITR, CMV enhancer, CBA promoter, SV40 small T intron, consensus Kozak sequence, PCCA coding sequence, SV40 polyadenylation signal sequence and 3' -ITR.

Protein localization:

propionyl-CoA carboxylase is a multimeric protein that localizes to the mitochondrial matrix (see Browner et al, (1989) Journal of Biological chemistry 264: 12680-5). Therefore, gene therapy vectors delivering PCCA and/or PCCB genes must be able to encode proteins that are functional and localized in the mitochondria. In one aspect, one or more of the viral vectors disclosed herein drive and regulate expression of a gene encoding a PCCA and/or PCCB protein located in the mitochondria (e.g., expression of PCCA or PCCB).

The pharmaceutical composition comprises:

the present disclosure provides compositions comprising the rAAV disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutical dosage forms suitable for administration of rAAV may be found, for example, in U.S. patent application publication No. 2012/0219528. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington pharmaceuticals (Remington's Pharmaceutical Sciences), e.w. martin, Mack Publishing co, Easton, Pa., 15 th edition (1975) describe compositions and dosage forms suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

As highlighted in the preceding paragraphs, the present disclosure relates in certain aspects to pharmaceutical compositions comprising the rAAV of the invention. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In exemplary embodiments, the pharmaceutical composition is formulated for intravenous administration.

In certain embodiments, the rAAV is formulated in a buffer/carrier suitable for infusion in a human subject. The buffer/carrier should contain components that prevent the rAAV from adhering to the infusion line, but do not interfere with the in vivo binding activity of the rAAV. Various suitable solutions may include one or more of the following: buffered saline, a surfactant, and a physiologically compatible salt or mixture of salts having an ionic strength adjusted to be equivalent to about 100mM sodium chloride (NaCl) to about 250mM sodium chloride or a physiologically compatible salt adjusted to an equivalent ionic concentration. The pH may be in the range of 6.5 to 8.5 or 7 to 8.5 or 7.5 to 8. Suitable surfactants or combinations of surfactants may be selected from poloxamers (i.e. triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene 10 (poly (oxypropylene)) and two hydrophilic chains of polyoxyethylene (poly (oxyethylene)) on both sides), SOLUTOL HS 15 (polyethylene glycol-15 hydroxystearate), LABRASOL (glyceryl polyoxyethylene octanoate), polyoxyethylene 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol.

Methods of treating propionic acidemia:

in another aspect, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to the human subject a therapeutically effective amount of at least one rAAV disclosed herein.

In one embodiment, the present disclosure provides a method of treating PA comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCA or an isoform or a functional fragment or variant thereof.

In another embodiment, the present disclosure provides a method of treating PA comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCB or an isoform or a functional fragment or variant thereof.

In yet another embodiment, the present disclosure provides a method of treating PA, the method comprising administering: (1) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence for PCCA or an isoform or a functional fragment or variant thereof; and (2) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of the coding sequence of PCCB or an isoform or a functional fragment or functional variant thereof. In certain embodiments, the rAAV of (1) and (2) may be administered simultaneously. In certain embodiments, the rAAV of (1) and (2) may be administered sequentially. In certain embodiments, the rAAV of (1) and (2) may be administered separately.

In certain embodiments, the present disclosure provides a method of treating PA, the method comprising administering: (1) a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCA and not PCCB, the promoter is a PCCA gene-specific endogenous promoter and not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence and not a PCCB gene-specific endogenous polyadenylation signal sequence; and/or (2) a rAAV comprising an AAV capsid and a vector genome, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the rAAV of (1) and (2) may be administered simultaneously. In certain embodiments, the rAAV of (1) and (2) may be administered sequentially. In certain embodiments, the rAAV of (1) and (2) may be administered separately.

In certain embodiments, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to a human subject diagnosed as having at least one mutation in PCCA a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCA or an isoform or functional fragment or functional variant thereof. In certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCA but not PCCB, the promoter is a PCCA gene-specific endogenous promoter but not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence but not a PCCB gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the mutation in PCCA is selected from table 1. In certain embodiments, the PCCA coding sequence is selected from SEQ ID NOs: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

In certain instances, the present disclosure provides a method of treating PA in a human subject, the method comprising administering to a human subject diagnosed as having at least one mutation in PCCB a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCB or an isoform or functional fragment or functional variant thereof. In certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence. In certain embodiments, the mutation in the PCCB is selected from table 2. In certain embodiments, the coding sequence of the PCCB is selected from the group consisting of SEQ ID NOs: 7-12.

In certain embodiments, the capsid is an AAV9 capsid.

A review article describing mutations in PCCA and PCCB that cause PA is provided in Ugarte et al, 1999, hum. Mutat.14(4): 275-.

In PA caused by mutated PCCA gene, the following mutations and polymorphisms in PCCA gene have been identified:

table 1-PCCA gene mutations and polymorphisms:

in PA caused by mutated PCCB genes, the following mutations and polymorphisms in the PCCB gene have been identified:

table 2-PCCB gene mutations and polymorphisms:

in certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed with at least one mutation selected from table 1 in PCCA, the method comprising administering to the human subject a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed with at least one mutation selected from table 1 in PCCA, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCA or an isoform or functional fragment or functional variant thereof. In certain embodiments, the PCCA coding sequence is selected from SEQ ID NOs: 1-6. In certain embodiments, the capsid is an AAV9 capsid.

In certain embodiments, the present disclosure provides a method of treating PA in a human subject diagnosed with at least one mutation selected from table 2 in PCCB, the method comprising administering to the human subject a therapeutically effective amount of at least one rAAV disclosed herein. In one embodiment, the present disclosure provides a method of treating PA in a human subject diagnosed with at least one mutation selected from table 2 in PCCB, the method comprising administering a rAAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises part or all of a coding sequence for PCCB or an isoform or functional fragment or functional variant thereof. In certain embodiments, the coding sequence of the PCCB is selected from the group consisting of SEQ ID NOs: 7-12. In certain embodiments, the capsid is an AAV9 capsid.

Any suitable method or route may be used to administer the rAAV or rAAV-containing compositions described herein. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In certain embodiments, the rAAV, a composition comprising a rAAV, or a composition comprising multiple raavs (e.g., one rAAV expressing PCCA and a second rAAV expressing PCCB) is administered intravenously.

The specific dose administered may be a uniform dose for each patient, e.g., 1.0x10 for each patient¹¹–1.0x10¹⁴Viral of Genomic Copies (GC). Alternatively, the dosage for a patient may be customized to the approximate weight or surface area of the patient. Other factors in determining an appropriate dosage may include the disease or disorder to be treated or prevented, the severity of the disease, the route of administration, the age, sex and medical condition of the patient. Further modifications of the calculations necessary to determine an appropriate therapeutic dose are routinely made by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosage can also be determined by using known assays for determining dosage used in conjunction with appropriate dose response data. The dosage for each patient may also be adjusted as the disease progression is monitored.

In certain embodiments, the rAAV is administered, e.g., at about 1.0x10¹¹Genome copy/kg patient body weight (GC/kg) to about 1x10¹⁴GC/kg, about 5X10¹¹Genome copy/kg patient body weight (GC/kg) to about 5x10¹³GC/kg or about 1X10¹²To about 1x10¹³Dosage of GC/kg, as measured by qPCR or digital droplet pcr (ddpcr). In certain embodiments, the rAAV is administered at about 1x10¹²To about 1x10¹³Genomic Copy (GC)/kg. In certain embodiments, the rAAV is administered at about 1.1x10¹¹About 1.3x10¹¹About 1.6x10¹¹About 1.9x10¹¹About 2x10¹¹About 2.5x10¹¹About 3.0x10¹¹About 3.5x10¹¹About 4.0x10¹¹About 4.5x10¹¹About 5.0x10¹¹About 5.5x10¹¹About 6.0x10¹¹About 6.5x10¹¹About 7.0x10¹¹About 7.5x10¹¹About 8.0x10¹¹About 8.5x10¹¹About 9.0x10¹¹About 9.5x10¹¹About 1.0x10¹²About 1.5x10¹²About 2.0x10¹²About 2.5x10¹²About 3.0x10¹²About 3.5x10¹²About 4.0x10¹²About 4.5x10¹²About 5.0x10¹²About 5.5x10¹²About 6.0x10¹²About 6.5x10¹²About 7.0x10¹²About 7.5x10¹²About 8.0x10¹²About 8.5x10¹²About 9.0x10¹²About 9.5x10¹²About 1.0x10¹³About 1.5x10¹³About 2.0x10¹³About 2.5x10¹³About 3.0x10¹³About 3.5x10¹³About 4.0x10¹³About 4.5x10¹³About 5.0x10¹³About 5.5x10¹³About 6.0x10¹³About 6.5x10¹³About 7.0x10¹³About 7.5x10¹³About 8.0x10¹³About 8.5x10¹³About 9.0x10¹³About 9.5x10¹³Genomic Copy (GC)/kg. The rAAV may be administered in a single dose or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10 doses or more) as needed to achieve the desired therapeutic result.

The agent may be administered one or more times per week, month or year, or even once every 2 to 20 years. For example, each dose may be administered at least 1 week apart, 2 weeks apart, 3 weeks apart, 1 month apart, 3 months apart, 6 months apart, or 1 year apart. One of ordinary skill in the art can readily estimate the repetition rate of administration based on the measured residence time and concentration of the targetable construct or complex in the body fluid or tissue.

Throughout this specification, where a composition is described as having, including, or comprising specific components, or where a process and method is described as having, including, or comprising specific steps, it is contemplated that there will additionally be present a composition of the invention consisting essentially of, or consisting of, the recited components, and that there will additionally be present a process and method of the invention consisting essentially of, or consisting of, the recited process steps.

In the present disclosure, where an element or component is described as being included in and/or selected from a recited list of elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from two or more of the recited elements or components.

Moreover, it should be understood that elements and/or features of the compositions or methods described herein may be combined in various different ways, whether explicitly or implicitly herein, without departing from the spirit and scope of the disclosure. For example, where a particular compound is referred to, the compound can be used in various embodiments of the compositions of the invention and/or in the methods of the invention, unless otherwise understood from the context. In other words, in the present disclosure, embodiments are described and depicted in a manner that enables a clear and concise application to be written and depicted, but it is to be understood and appreciated that various different combinations or separations of embodiments may be made without departing from the teachings and inventions herein. For example, it should be recognized that all of the features described and depicted herein may be applicable to all of the aspects of the invention described and depicted herein.

It is to be understood that at least one of the expressions "… …" includes each recited subject following the expression and various different combinations of two or more of the recited subjects, respectively, unless otherwise understood from the context and usage. The expression "and/or" in relation to three or more of the recited subjects shall be understood to have the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," "including," and "containing," including grammatical equivalents thereof, is to be generally understood as open-ended and non-limiting, e.g., without excluding additional unrecited elements or steps, unless specifically stated otherwise or otherwise understood from context.

Where the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" means within ± 10% of the nominal value, unless otherwise indicated or inferred.

It will be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Examples

The present disclosure now being generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure in any way.

Example 1: RNA and protein expression of PCCA

RNA expression

This example relates to RNA expression of PCCA as determined by RT-qPCR after transfection of a continuous liver cell line (HepG 2). Simply comeStated, HepG2 cells were transfected with a control plasmid (plasmid with empty vector, no TX), a rAAV vector plasmid with nucleotide sequences encoding enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with nucleotide sequences encoding the complete native sequence of human PCCA (DTC346), or a rAAV vector plasmid with nucleotide sequences encoding codon optimized sequence of human PCCA (DTC347, DTC348, or DTC349) using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. HepG2 cells were harvested 72 hours post transfection and lysed. RNA was extracted from lysed HepG2 cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were measured by OD260/280 and OD260/230 ratios using NanoDrop ND-1000(Thermo Scientific). Total RNA was reverse transcribed using the high-capacity cDNA reverse transcription kit (ThermoFisher Scientific) according to the manufacturer's instructions. Real-time RT-qPCR was performed using an Applied Biosystems 7500 real-time PCR system using Power SYBR Green PCR master mix (Applied Biosystems) following the manufacturer's instructions. By delta-delta Ct (2)^-ΔΔCt) Methods calculate fold changes in PCCA expression using RNA polymerase II polypeptide a (POLR2A) as an internal control for each sample and normalize the calculated fold changes to a "no TX" control (see Livak KJ, Schmittgen TD (2001), "Analysis of relative gene expression using real-time quantitative PCR and the 2(-delta C (T)) method" (Methods of relative gene expression using real-time quantitative PCR and the 2(-delta C (T)), Methods 25(4): 402-. FIG. 3 is a bar graph showing the fold change in PCCA RNA expression determined by RT-qPCR after transfection of continuous liver cell lines (HepG2) with control plasmids (plasmids with empty vector, without TX), a rAAV vector plasmid with a nucleotide sequence encoding the enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the entire native sequence of human PCCA (DTC346), or a rAAV vector plasmid with a nucleotide sequence encoding the codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC 349).

The plotted data shows that RNA expression of fully native human PCCA after transfection with DTC346 is comparable to the expression of the two codon-optimized versions of human PCCA after transfection with DTC347 and DTC 348. Cells transfected with DTC349 exhibited higher RNA expression.

Protein expression

This example also relates to protein expression of PCCA following transfection with the continuous liver cell lines HepG2 and Huh 7. Briefly, HepG2 and Huh7 cell lines were transfected with a control plasmid (plasmid with empty vector, no TX), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA (DTC346), a rAAV vector plasmid with a nucleotide sequence encoding codon-optimized sequence of human PCCA (DTC347, DTC348 or DTC349), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA and a nucleotide sequence encoding the complete native sequence of human PCCB (DTC365) or a rAAV vector plasmid with a nucleotide sequence encoding the enhanced green fluorescent protein eGFP (DTC343) using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. Cells were harvested 72 hours post transfection and lysed with NP-40 lysis buffer (50mM Tris & HCl pH8.0, 150mM NaCl, 1.0% NP-40) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail 2+3(Sigma P5726 and P0044). Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to

Polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Western blot analysis was performed using 1:1000 dilution of anti-PCCA (Abcam, Cambridge, UK) antibody followed by a Secondary antibody (Licor IRDye Secondary Antibodies) conjugated to a far-red fluorophore.

Fig. 4A is a photograph showing protein expression levels detected by Western blotting in continuous liver cell lines (HepG2) transfected with a control plasmid (a plasmid with an empty vector, without TX), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA (DTC346), a rAAV vector plasmid with a nucleotide sequence encoding a codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC349), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA and a nucleotide sequence encoding the complete native sequence of human PCCB (DTC365), or a rAAV vector plasmid with a nucleotide sequence encoding enhanced green fluorescent protein eGFP (DTC343), respectively, from lane 1 to lane 7. Beta-actin ("actin") was used as a loading control.

As shown in figure 4A, there was an expected basal level of PCCA protein expression in the "no TX" control (lane 1), and there was overexpression of PCCA protein in cells transfected with DTC346 (lane 2) and cells transfected with DTC347 (lane 3) or DTC348 (lane 4) when compared to the "no TX" control (lane 1). Cells transfected with DTC349 (lane 5) did not show overexpression of PCCA protein, although this version was observed to express large amounts of RNA. As expected, cells transfected with DTC343 (lane 7) showed eGFP expression.

Fig. 4B is a photograph showing the protein expression level detected by Western blotting in serial hepatocyte lines (Huh7) transfected with a control plasmid (a plasmid with an empty vector without TX), a rAAV vector plasmid with a nucleotide sequence encoding the enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA (DTC346), a rAAV vector plasmid with a nucleotide sequence encoding the codon-optimized sequence of human PCCA (DTC347, DTC348 or DTC349), or a rAAV vector plasmid with a nucleotide sequence encoding the complete native sequence of human PCCA and a nucleotide sequence encoding the complete native sequence of human PCCB (DTC365), respectively, from lane 1 to lane 7. Beta-actin ("actin") was used as a loading control.

As shown in figure 4B, there was an expected basal level of PCCA protein expression in the "no TX" control (lane 1), and there was overexpression of PCCA protein in cells transfected with DTC346 (lane 3) and cells transfected with DTC347 (lane 4) or DTC348 (lane 5) when compared to the "no TX" control (lane 1). Cells transfected with DTC349 (lane 6) did not show overexpression of PCCA protein, although this version was observed to express large amounts of RNA. As expected, cells transfected with DTC343 (lane 2) showed eGFP expression.

Example 2: localization of overexpressed human PCCA protein to mitochondria

This example shows that human PCCA overexpressed in a continuous liver cell line (HepG2) is localized to mitochondria. Briefly, a continuous liver cell line (HepG2) was transfected with a rAAV vector plasmid (DTC346) carrying a nucleotide sequence encoding the entire native sequence of human PCCA. After transfection, the cells were subjected to subcellular fractionation, in which the cytoplasmic and mitochondrial fractions were separated. Whole cell lysates were also processed. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to

Polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Western blot analysis was performed using 1:1000 dilution of anti-PCCA (Abcam, Cambridge, UK) antibody followed by a Secondary antibody (Licor IRDye Secondary Antibodies) conjugated to a far-red fluorophore. FIG. 5 is a photograph showing the levels of PCCA protein expression detected by Western blotting in Whole Cell Lysates (WCL), subcellular fractions (cytosolic fraction (cyto) and mitochondrial fractions (mitochondria)) isolated from continuous liver cell lines (HepG2) transfected with or without a rAAV vector plasmid (DTC346) carrying a nucleotide sequence encoding the entire native sequence of human PCCA. 1X and 2X represent the relative volumes of the loaded mitochondrial fractions. Beta-actin ("actin") was used as a loading control.

As shown in figure 5 and consistent with the results shown in figures 4A-B, cells transfected with a rAAV vector plasmid (DTC346) bearing a nucleotide sequence encoding the entire native sequence of human PCCA showed PCCA overexpression in whole cell lysates when compared to untransfected control cells (lane 7 versus lane 8 in figure 5). The level of a protein known to localize to mitochondria, mitofolin, was investigated to determine the relative purity of isolated cell fractions. As shown in figure 5, the cytosolic fraction of untransfected and DTC 346-transfected cells did not show detectable levels of mitofil (lane 1 compared to lane 2 in figure 5), whereas as expected mitofil was readily detected in the mitochondrial fraction and whole cell lysates (lanes 3-8 in figure 5). The over-expressed PCCA was mainly localized to mitochondria (lane 2 in fig. 5 compared to lanes 4 and 6).

Example 3: comparison of PCCA AAV9 Titer production

This example relates to a comparison of PCCA AAV9 titer production obtained from rAAV vector plasmids (DTC346) with nucleotide sequences encoding the entire native sequence of human PCCA or rAAV vector plasmids (DTC347, DTC348, or DTC349) with nucleotide sequences encoding codon-optimized sequences of human PCCA. Triple transfection of a continuous kidney cell line (HEK293) with a rAAV vector plasmid (DTC346) with a nucleotide sequence encoding the complete native sequence of human PCCA, a rAAV vector plasmid (DTC347, DTC348 or DTC349) with a nucleotide sequence encoding a codon-optimized sequence of human PCCA, or a rAAV vector plasmid (DTC343) with a nucleotide sequence encoding an enhanced green fluorescent protein eGFP; each of the vector plasmids was expressed in cells co-transfected with a plasmid expressing the AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral helper functions (pAdHelper). Untransfected cells were used as a control and are denoted "no TX" in fig. 6. Cell supernatants were harvested and treated with DNase I or equivalent to eliminate non-capsid coated DNA and to facilitate quantification of DNase Resistant Particle (DRP) titers using primer/probe sets specific for polyadenylation signals included in the design of these constructs. Figure 6 is a bar graph showing, from left to right, the fold change in PCCA AAV9 titers (in Genomic Copies (GC)/mL) determined by qPCR after triple transfection of a continuous renal cell line (HEK293) transfected with a rAAV vector plasmid (DTC343) with nucleotide sequences encoding the enhanced green fluorescent protein eGFP, a rAAV vector plasmid (DTC346) with nucleotide sequences encoding the complete native sequence of human PCCA, or a rAAV vector plasmid (DTC347, DTC348, or DTC349) with nucleotide sequences encoding codon-optimized sequences of human PCCA, each plasmid being expressed in cells co-transfected with a plasmid expressing the AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral helper functions (pAdHelper). Untransfected cells were used as controls and were denoted "without TX". Figure 6 shows fold change in PCCA AAV9 titers (in Genomic Copies (GC)/ml) relative to untransfected cells as determined by qPCR. The data shown in figure 6 demonstrate comparable rAAV titers between constructs expressing native, intact human PCCA (DTC346) and all codon optimized versions (DTC347, DTC348 and DTC 349).

Example 4: testing PCCA DNA integrity

After production of AAV9 rAAV virions in HEK293 cells by triple transfection, cell supernatants were harvested and incubated with AAVX resin (Thermo Fisher) or equivalent. AAV9 rAAV virions were purified and then concentrated using Amicon centrifugal tube filters (Millipore Sigma). The purified virus was incubated in alkaline lysis buffer, loaded into DNA agarose gel, and run overnight in the presence of alkaline running buffer. Fig. 7 is a photograph of DNA alkaline sepharose from left to right of AAV9 particles of affinity-purified AAV (raav) vector cassettes containing a recombinant AAV (raav) vector cassette expressing PCCA and PCCB (DTC365), a recombinant AAV (raav) vector cassette expressing PCCA (DTC349, DTC348, DTC347 or DTC346), or a recombinant AAV (raav) vector cassette expressing eGFP (DTC343), respectively.

As shown in fig. 7, the gel photographs show clear single bands at the expected size of the native and codon optimized PCCA vector cassette, indicating proper packaging of the vector genome (DTC346, DTC347, DTC348 and DTC 349). The eGFP control (DTC343) indicated packaging of the two bands, consistent with known limitations of the packaged vectorette genome.

Example 5: RNA and protein expression of PCCB

RNA expression

This example relates to RNA expression of PCCB as determined by RT-qPCR after transfection of a continuous liver cell line (HepG 2). Briefly, HepG2 cells were transfected with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmid with nucleotide sequences encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmid with nucleotide sequences encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmid with nucleotide sequences encoding codon optimized sequence of human PCCB (DTC367, DTC370, DTC368, DTC369, or DTC371) using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. HepG2 cells were harvested 72 hours post transfection and lysed. RNA was extracted from lysed HepG2 cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were measured by OD260/280 and OD260/230 ratios using NanoDrop ND-1000(Thermo Scientific). Total RNA was reverse transcribed using the high-capacity cDNA reverse transcription kit (ThermoFisher Scientific) according to the manufacturer's instructions. Real-time RT-qPCR was performed using an Applied Biosystems 7500 real-time PCR system using Power SYBR Green PCR master mix (Applied Biosystems) following the manufacturer's instructions. By delta-delta Ct (2)^-ΔΔCt) Methods calculate fold changes in PCCB expression using RNA polymerase II polypeptide A (POLR2A) as an internal control for each sample and normalizing the calculated fold changes to a "no TX" control (see Livak KJ, Schmittgen TD (2001), "Analysis of relative gene expression using real-time quantitative PCR and the 2(-delta C (T)) method" (Methods of relative gene expression using real-time quantitative PCR and the 2(-delta C (T)), Methods 25(4): 402-. Figure 8 is a bar graph showing fold changes in PCCB RNA expression determined by RT-qPCR after transfection of continuous liver cell lines (HepG2) with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmid with nucleotide sequences encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmid with nucleotide sequences encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmid with nucleotide sequences encoding codon-optimized sequences of human PCCB (DTC367, DTC370, DTC368, DTC369, or DTC 371).

The plotted data shows that the fully native list of RNA expression of human PCCB after transfection with DTC366 is comparable to the expression of the four codon optimized versions of human PCCB after transfection with DTC367, DTC368, DTC370, or DTC 371. Cells transfected with DTC369 showed lower RNA expression.

Protein expression

This example also relates to protein expression of PCCB following transfection with the continuous liver cell lines HepG2 and Huh 7. Briefly, Lipofectamine 2000(Invitrogen) was used, as will be described in the manufacturer's instructionsHepG2 and Huh7 cell lines were transfected with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmids with nucleotide sequences encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmids with nucleotide sequences encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmids with nucleotide sequences encoding codon optimized sequences of human PCCB (DTC367, DTC368, DTC369, DTC370, or DTC 371). Cells were harvested 72 hours post transfection and lysed with NP-40 lysis buffer (50mM Tris & HCl pH8.0, 150mM NaCl, 1.0% NP-40) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail 2+3(Sigma P5726 and P0044). Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel and transferred to IMMUN-

Polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Western blot analysis was performed using 1:1000 dilution of anti-PCCB (Abcam, Cambridge, UK) antibody followed by a Secondary antibody (Licor IRDye Secondary Antibodies) conjugated to a far-red fluorophore.

Fig. 9A is a photograph showing the level of protein expression detected by Western blotting in continuous liver cell lines (HepG2) transfected with a control plasmid (plasmid with empty vector, no TX), a rAAV vector plasmid with a nucleotide sequence encoding an enhanced green fluorescent protein eGFP (DTC343), a rAAV vector plasmid with a nucleotide sequence encoding the entire native sequence of human PCCB (DTC366), or a rAAV vector plasmid with a nucleotide sequence encoding a codon-optimized sequence of human PCCB (DTC367, DTC368, DTC369, DTC370, or DTC371), respectively, from lane 1 to lane 8. Beta-actin ("actin") was used as a loading control.

As shown in fig. 9A, there was an expected basal level of PCCB protein expression in the "no TX" control (lane 1), and there was overexpression of PCCB protein in cells transfected with DTC366 (lane 3) and cells transfected with DTC367 (lane 4) or DTC370 (lane 7) when compared to the "no TX" control (lane 1). Cells transfected with DTC368 (lane 5), DTC369 (lane 6) or DTC371 (lane 8) showed no overexpression of PCCB protein. As expected, cells transfected with DTC343 (lane 2) showed eGFP expression.

Fig. 9B is a photograph showing the level of protein expression detected by Western blotting in serial hepatocyte lines (Huh7) transfected with control plasmids (plasmids with empty vector, no TX), rAAV vector plasmid with nucleotide sequence encoding enhanced green fluorescent protein eGFP (DTC343), rAAV vector plasmid with nucleotide sequence encoding the complete native sequence of human PCCB (DTC366), or rAAV vector plasmid with nucleotide sequence encoding codon optimized sequence of human PCCB (DTC367, DTC369, DTC368, DTC370, or DTC371), respectively, from lane 1 to lane 8. Beta-actin ("actin") was used as a loading control.

As shown in fig. 9B, there was an expected basal level of PCCB protein expression in the "no TX" control (lane 1), and there was overexpression of PCCB protein in cells transfected with DTC366 (lane 3) and cells transfected with DTC367 (lane 4) or DTC370 (lane 7) when compared to the "no TX" control (lane 1). Cells transfected with DTC368 (lane 6), DTC369 (lane 5) or DTC371 (lane 8) showed no overexpression of PCCB protein. As expected, cells transfected with DTC343 (lane 2) showed eGFP expression.

Example 6: localization of overexpressed human PCCB proteins to mitochondria

This example shows that human PCCB overexpressed in a continuous liver cell line (HepG2) is localized to mitochondria. Briefly, a continuous liver cell line (HepG2) was transfected with a rAAV vector plasmid (DTC366) carrying a nucleotide sequence encoding the entire native sequence of human PCCB. After transfection, the cells were subjected to subcellular fractionation, in which the cytoplasmic and mitochondrial fractions were separated. Whole cell lysates were also processed. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to

Polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Western blot analysis Using 1:1000 dilution of anti-PCCB (Abcam, Cambri)dge, UK), and then using a Secondary antibody (Licor IRDye Secondary Antibodies) conjugated to a far-red fluorophore.

Figure 10 is a photograph showing the levels of PCCB protein expression detected by Western blot in Whole Cell Lysates (WCL), subcellular fractions (cytosolic fraction (cyto) and mitochondrial fractions (mitochondria)) isolated from continuous liver cell lines (HepG2) transfected with or without a rAAV vector plasmid (DTC366) carrying the nucleotide sequence encoding the entire native sequence of human PCCB. 1X and 2X represent the relative volumes of the loaded mitochondrial fractions. Beta-actin ("actin") was used as a loading control.

As shown in figure 10 and consistent with the foregoing results, cells transfected with a rAAV vector plasmid (DTC366) with a nucleotide sequence encoding the entire native sequence of human PCCB exhibited PCCB protein overexpression in whole cell lysates when compared to untransfected control cells (lane 7 versus lane 8 in figure 10). The level of a protein known to localize to mitochondria, mitofolin, was investigated to determine the relative purity of isolated cell fractions. As shown in figure 10, the cytosolic fraction of untransfected and DTC 366-transfected cells did not show detectable levels of mitofil (lane 1 compared to lane 2), while as expected, mitofil was readily detected in the mitochondrial fraction and whole cell lysates (lanes 3-8). The overexpressed PCCBs were mainly localized to mitochondria (lane 2 compared to lanes 4 and 6).

Example 7: comparison of PCCB AAV9 Titer yields

This example relates to comparison of PCCB AAV9 titer yields obtained from rAAV vector plasmids (DTC366) with nucleotide sequences encoding the entire native sequence of human PCCB or rAAV vector plasmids (DTC367, DTC368, DTC369, DTC370, or DTC371) with nucleotide sequences encoding codon-optimized sequences of human PCCB. Triple transfection of a continuous kidney cell line (HEK293) with a rAAV vector plasmid (DTC343) with a nucleotide sequence encoding an enhanced green fluorescent protein eGFP, a rAAV vector plasmid (DTC366) with a nucleotide sequence encoding the complete native sequence of human PCCB, or a rAAV vector plasmid (DTC367, DTC368, DTC369, DTC370, or DTC371) with a nucleotide sequence encoding a codon-optimized sequence of human PCCA; each of the vector plasmids was expressed in cells co-transfected with a plasmid expressing the AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral helper functions (pAdHelper). Untransfected cells were used as a control and are denoted "no TX" in fig. 11. Cell supernatants were harvested and treated with DNase I or equivalent to eliminate non-capsid coated DNA and to facilitate quantification of DNase Resistant Particle (DRP) titers using primer/probe sets specific for polyadenylation signals included in the design of these constructs. Figure 11 is a bar graph showing, from left to right, the fold change in PCCB AAV9 titers (in Genomic Copies (GC)/mL) determined by qPCR after triple transfection of a continuous renal cell line (HEK293) transfected with a rAAV vector plasmid (DTC343) with nucleotide sequences encoding the enhanced green fluorescent protein eGFP, a rAAV vector plasmid (DTC366) with nucleotide sequences encoding the complete native sequence of human PCCB, or a rAAV vector plasmid (DTC367, DTC368, DTC369, DTC370, or DTC371) with nucleotide sequences encoding codon-optimized sequences of human PCCA, each plasmid being expressed in cells co-transfected with a plasmid expressing AAV9 capsid (pAAV2/9) and a plasmid providing adenoviral-assisted function (pAdHelper). Untransfected cells were used as controls and were denoted "without TX". Figure 11 shows fold change in PCCB AAV9 titers (in Genomic Copies (GC)/ml) relative to untransfected cells as determined by qPCR. The data shown in figure 11 demonstrates comparable rAAV titers between the construct expressing intact human PCCB of origin (DTC366) and all codon optimized versions (DTC367, DTC368, DTC369, DTC370, and DTC 371).

Example 8: testing PCCB DNA integrity

After production of AAV9 rAAV virions in HEK293 cells by triple transfection, cell supernatants were harvested and incubated with AAVX resin (Thermo Fisher) or equivalent. AAV9 rAAV virions were purified and then concentrated using Amicon centrifugal tube filters (Millipore Sigma). The purified virus was incubated in alkaline lysis buffer, loaded into DNA agarose gel, and run overnight in the presence of alkaline running buffer. Fig. 12 is a photograph of DNA alkaline agarose gels from left to right of affinity purified AAV9 particles containing a control plasmid (plasmid with empty vector, no TX), a recombinant AAV (raav) vector cassette expressing eGFP (DTC343), or a recombinant AAV (raav) vector cassette expressing PCCB (DTC366, DTC367, DTC368, DTC369, DTC370, or DTC371), respectively.

As shown in fig. 12, the gel photographs show clear single bands at the expected size of the native and codon optimized PCCB vector cassette, indicating suitable packaging of the vector genome (DTC366, DTC367, DTC368, DTC369, DTC370, and DTC 371). The eGFP control (DTC343) indicated packaging of the two bands, consistent with known limitations of the packaged vectorette genome.

Example 9: administration of PCCA viral vectors to wild-type mice

This example relates to the expression of human PCCA protein in wild-type FVB mice following treatment with rAAV expressing intact native human PCCA (DTC346) or codon-optimized (DTC347, DTC348 and DTC349) human PCCA. Briefly, mice (n-2 or 3) were treated with 1.66 × 10¹¹Viral Genome (VG) or 5x10¹¹Expression of VGs is represented by SEQ ID NO: 38, or raavs expressing codon-optimized human PCCA (DTC347, DTC348, and DTC 349). Human PCCA protein and endogenous mouse PCCA protein were detected by LC-MS. Figure 13 is a bar graph showing the percentage of human PCCA protein expression relative to mouse endogenous PCCA expression in a wild type FVB mouse following rAAV treatment with either the complete native sequence encoding human PCCA (DTC346) or the codon optimized sequence of human PCCA (DTC347, DTC348 or DTC 349). Error bars indicate standard deviation. The data display presented in FIG. 13, from 1.66x10¹¹Administration of native PCCA of VG (DTC346) human PCCA expressed 2.5% of endogenous mouse PCCA levels and ranged from 5x10¹¹Administration of native PCCA viral vector of VG (DTC346) expressed human PCCA at 11.6% of endogenous mouse PCCA levels.

Example 10: human PCCA activity in a mouse model of a sub-allele PCCA

This example relates to testing humans in a mouse model of the sub-effective allele PCCAPCCA activity. Deletion of the PCCA gene in mice mimics the most severe form of the human disease. PCCA gene-deleted mice died within 36 hours after birth, making it difficult to test intravenous systemic therapy in them (see Guenzel et al, Mol ther.2013 Jul; 21(7): 1316-. Because of the introduction of transgenes encoding human PCCA with the pathogenic a138T mutation in the background of mouse PCCA knockouts, the hypo-allelic PCCA mice have reduced PCCA activity. This mutated PCCA gene results in elevated levels of propionyl-carnitine, methyl citrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy, similar to the levels of these compounds in patients with Propionic Acidemia (PA). Briefly, mice (n ═ 5) were treated with 1.66 × 10¹¹Viral Genome (VG) or 5x10¹¹The VG is encoded by SEQ ID NO: a rAAV of the complete native sequence of human PCCA (DTC346) shown at 38. PBS treated mice were used as control mice.

PCCA expression level and enzyme activity assay

At 6 weeks post-injection, mouse livers were homogenized and human wild-type PCCA protein was measured by LC-MS. Figure 14 is a bar graph showing the concentration (in nmol/g protein) of human wild-type PCCA protein expressed in a mouse model of sub-allele PCCA (a138T mutant) following rAAV treatment with a rAAV encoding the complete native sequence of human PCCA (DTC346) or a codon-optimized sequence of human PCCA (DTC347, DTC348, or DTC 349). The percentages listed represent human wild-type PCCA expression calculated as a percentage of endogenous PCCA protein levels relative to wild-type FVB mice. Error bars indicate standard deviation. The percentages listed represent human wild-type PCCA expression calculated as a percentage of endogenous PCCA protein levels relative to wild-type FVB mice. Consistent with the foregoing results, human PCCA protein expressed from a rAAV encoding codon-optimized human PCCA exhibits lower expression levels when compared to native human PCCA protein expressed from a rAAV encoding the entire native human PCCA. PCCA enzyme activity of liver homogenates was measured as sodium bicarbonate [ C14] incorporation using a scintillation counter. Figure 15 is a bar graph of human PCCA activity as measured by mean Count Per Minute (CPM) data in a mouse model of subthreshold allele PCCA (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). Error bars indicate standard deviation. The percentages listed describe CPM data relative to that observed in wild-type FVB mice. Indicates p <0.01 compared to PBS treated group using Dunnett's multiple comparison test.

As shown in fig. 15, with higher dose (5x 10)¹¹VG) encoding the entire native human PCCA, showed a statistically significant increase (11%) in PCCA activity compared to PBS-treated mice.

Effect of administration of native PCCA Virus vector to Subsequent allele PCCA mice on the concentration of known biomarkers of Propioniemia (PA)

This example also relates to testing the effect of administering rAAV (DTC346), encoding the complete native sequence of human PCCA, to sub-allele PCCA mice on plasma concentrations of known biomarkers of PA. Briefly, mice (n ═ 5) were treated with 1.66 × 10¹¹VG or 5x10¹¹The VG is encoded by SEQ ID NO: a rAAV (DTC346) containing the complete native sequence of human PCCA as shown in item 38 is injected intravenously. Plasma concentrations of the known biomarkers of PA (propionyl-carnitine (C3), acetyl-carnitine (C2) and 2-methylcitric acid (2MC)) were determined by liquid chromatography-mass spectrometry (LC-MS). Figures 16A-C are bar graphs showing the plasma concentrations of known propionic acid blood biomarkers in a murine model of suballet allele PCCA (a138T mutant) before (Pre) and 2, 3, 4, and 6 weeks (2W, 3W, 4W, and 6W, respectively) after treatment with rAAV (DTC346) encoding the complete native sequence of human PCCA. Error bars indicate standard deviation. Denotes p compared to PBS treatment group<0.01, p represents the p compared to the PBS treated group<0.001 using Dunnett's multiple comparison test. Figure 16A is a bar graph showing plasma concentrations of C3 (propionyl carnitine) in a mouse model of sub-effective allele PCCA (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346). FIG. 16B is a bar graph of plasma C3/C2 concentration ratios (propionyl-carnitine/acetyl-carnitine) in a mouse model of sub-effective allele PCCA (A138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC346). Figure 16C is a bar graph showing plasma concentrations of 2-methyl citrate (2MC) in a sub-allele PCCA mouse model (a138T mutant) following treatment with rAAV encoding the complete native sequence of human PCCA (DTC 346).

With either dose (1.66X 10)¹¹VG or 5x10¹¹VG) encoding the entire native sequence of human PCCA, rAAV (DTC346) treated mice showed statistically significant reductions in plasma C3, C3/C2, and 2-MC levels compared to PBS treated mice.

Numbering implementation

Embodiments disclosed herein include embodiments P1 through P136 provided in the numbered embodiments of the present disclosure.

Embodiment P1: a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising, in 5 'to 3' order:

(a) a 5' ITR sequence;

(b) a promoter sequence;

(c) a partial or complete coding sequence for PCCA; and

(d) 3' ITR sequence.

Embodiment P2: the rAAV according to embodiment P1, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10, or hu 37.

Embodiment P3: the rAAV according to embodiment P2, wherein the AAV capsid is from AAV 9.

Embodiment P4: the rAAV according to embodiment P2, wherein the AAV capsid is from AAV 8.

Embodiment P5: the rAAV according to embodiment P1, wherein the AAV capsid is an AAV9 variant capsid.

Embodiment P6: the rAAV according to any one of embodiments P1-P5, wherein the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a Cytomegalovirus (CMV) immediate early gene promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, and a CAG promoter.

Embodiment P7: the rAAV according to embodiment P6, wherein the promoter is a CBA promoter.

Embodiment P8: the rAAV according to any one of embodiments P1 to P7, wherein part or all of the coding sequence of the PCCA is a wild-type coding sequence.

Embodiment P9: the rAAV according to embodiment P8, wherein the coding sequence for PCCA comprises SEQ ID NO: 1.

embodiment P10: the rAAV according to any one of embodiments P1 to P7, wherein the partial or complete coding sequence of PCCA is a codon optimized coding sequence.

Embodiment P11: the rAAV according to embodiment P10, wherein the coding sequence for PCCA comprises a sequence selected from SEQ ID NOs: 2-6.

Embodiment P12: the rAAV according to any one of embodiments P1 to P11, wherein the 5' ITR sequence is from AAV 2.

Embodiment P13: the rAAV according to any one of embodiments P1 to P11, wherein the 3' ITR sequence is from AAV 2.

Embodiment P14: the rAAV according to any one of embodiments P1 to P11, wherein the 5 'ITR and 3' ITR sequences are from AAV 2.

Embodiment P15: the rAAV according to any one of embodiments P12 to P14, wherein the 5 'ITR and 3' ITR sequences comprise SEQ ID NOs: 15 or consist thereof.

Embodiment P16: the rAAV according to any one of embodiments P1 to P11, wherein the 5 'ITR sequence and/or 3' ITR sequence are from a non-AAV 2 source.

Embodiment P17: the rAAV according to any one of embodiments P1 to P16, wherein the packaged genome further comprises one or more enhancer sequences.

Embodiment P18: the rAAV according to embodiment P17, wherein the enhancer is selected from the group consisting of a cytomegalovirus immediate early gene (CMV) enhancer, a transthyretin enhancer (enTTR), a chicken β -actin (CBA) enhancer, an En34 enhancer, and an ApoE enhancer.

Embodiment P19: the rAAV according to embodiment P18, wherein the enhancer is a CMV enhancer.

Embodiment P20: the rAAV according to embodiment P19, wherein the enhancer comprises the amino acid sequence of SEQ ID NO: 19 or consist thereof.

Embodiment P21: the rAAV according to any one of embodiments P18 to P20, wherein the enhancer is located upstream of the promoter sequence.

Embodiment P22: the rAAV according to any one of embodiments P1-P21, wherein the packaged genome further comprises one or more intron sequences.

Embodiment P23: the rAAV according to embodiment P22, wherein the intron is selected from the group consisting of the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, and the hFIX intron.

Embodiment P24: the rAAV according to embodiment P23, wherein the intron is the SV40 small T intron.

Embodiment P25: the rAAV according to embodiment P24, wherein the intron comprises the sequence of SEQ ID NO: 20 or consist thereof.

Embodiment P26: the rAAV according to embodiment P23, wherein the intron is a rHBB intron.

Embodiment P27: the rAAV according to embodiment P26, wherein the intron comprises the sequence of SEQ ID NO: 21 or consist thereof.

Embodiment P28: the rAAV according to any one of embodiments P1-P27, wherein the packaged genome further comprises a polyadenylation signal sequence.

Embodiment P29: the rAAV according to embodiment P28, wherein the polyadenylation signal sequence is selected from a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, and a rabbit β globin polyadenylation signal sequence.

Embodiment P30: the rAAV according to embodiment P29, wherein the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence.

Embodiment P31: the rAAV according to embodiment P30, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 22 or consist thereof.

Embodiment P32: the rAAV according to embodiment P29, wherein the polyadenylation signal sequence is an SV40 polyadenylation signal sequence.

Embodiment P33: the rAAV according to embodiment P32, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 23 or consist thereof.

Embodiment P34: a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising, in 5 'to 3' order:

(a) a 5' ITR sequence;

(b) a promoter sequence;

(c) a partial or complete coding sequence of PCCB; and

(d) 3' ITR sequence.

Embodiment P35: the rAAV according to embodiment P34, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10, or hu 37.

Embodiment P36: the rAAV according to embodiment P35, wherein the AAV capsid is from AAV 9.

Embodiment P37: the rAAV according to embodiment P35, wherein the AAV capsid is from AAV 8.

Embodiment P38: the rAAV according to embodiment P34, wherein the AAV capsid is an AAV9 variant capsid.

Embodiment P39: the rAAV according to any one of embodiments P34-P38, wherein the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a cytomegalovirus immediate early gene (CMV) promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, and a CAG promoter.

Embodiment P40: the rAAV according to embodiment P39, wherein the promoter is a CBA promoter.

Embodiment P41: the rAAV according to any one of embodiments P34 to P40, wherein part or all of the coding sequence of the PCCB is a wild-type coding sequence.

Embodiment P42: the rAAV according to embodiment P41, wherein the coding sequence of PCCB comprises SEQ ID NO: 7.

embodiment P43: the rAAV according to any one of embodiments P34 to P40, wherein part or all of the coding sequence of the PCCB is a codon optimized coding sequence.

Embodiment P44: the rAAV according to embodiment P43, wherein the coding sequence of the PCCB comprises a sequence selected from SEQ ID NOs: 8-12.

Embodiment P45: the rAAV according to any one of embodiments P34 to P44, wherein the 5' ITR sequence is from AAV 2.

Embodiment P46: the rAAV according to any one of embodiments P34 to P44, wherein the 3' ITR sequence is from AAV 2.

Embodiment P47: the rAAV according to any one of embodiments P34 to P44, wherein the 5 'ITR and 3' ITR sequences are from AAV 2.

Embodiment P48: the rAAV according to any one of embodiments P45 to P47, wherein the 5 'ITR and 3' ITR sequences comprise SEQ ID NOs: 15 or consist thereof.

Embodiment P49: the rAAV according to any one of embodiments P34 to P44, wherein the 5 'ITR sequence and/or 3' ITR sequence are from a non-AAV 2 source.

Embodiment P50: the rAAV according to any one of embodiments P34 to P49, wherein the packaged genome further comprises one or more enhancer sequences.

Embodiment P51: the rAAV according to embodiment P50, wherein the enhancer is selected from the group consisting of a cytomegalovirus immediate early gene (CMV) enhancer, a transthyretin enhancer (enTTR), a chicken β -actin (CBA) enhancer, an En34 enhancer, and an ApoE enhancer.

Embodiment P52: the rAAV according to embodiment P51, wherein the enhancer is a CMV enhancer.

Embodiment P53: the rAAV according to embodiment P52, wherein the enhancer comprises the amino acid sequence of SEQ ID NO: 19 or consist thereof.

Embodiment P54: the rAAV according to any one of embodiments P51 to P53, wherein the enhancer is located upstream of the promoter sequence.

Embodiment P55: the rAAV according to any one of embodiments P34-P54, wherein the packaged genome further comprises one or more intron sequences.

Embodiment P56: the rAAV according to embodiment P55, wherein the intron is selected from the group consisting of the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, and the hFIX intron.

Embodiment P57: the rAAV according to embodiment P56, wherein the intron is the SV40 small T intron.

Embodiment P58: the rAAV according to embodiment P57, wherein the intron comprises the sequence of SEQ ID NO: 20 or consist thereof.

Embodiment P59: the rAAV according to embodiment P56, wherein the intron is a rHBB intron.

Embodiment P60: the rAAV according to embodiment P59, wherein the intron comprises the sequence of SEQ ID NO: 21 or consist thereof.

Embodiment P61: the rAAV according to any one of embodiments P34-P60, wherein the packaged genome further comprises a polyadenylation signal sequence.

Embodiment P62: the rAAV according to embodiment P61, wherein the polyadenylation signal sequence is selected from a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, and a rabbit β globin polyadenylation signal sequence.

Embodiment P63: the rAAV according to embodiment P62, wherein the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence.

Embodiment P64: the rAAV according to embodiment P63, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 22 or consist thereof.

Embodiment P65: the rAAV according to embodiment P62, wherein the polyadenylation signal sequence is an SV40 polyadenylation signal sequence.

Embodiment P66: the rAAV according to embodiment P65, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 23 or consist thereof.

Embodiment P67: a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising as operably linked components in 5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) a partial or complete coding sequence for PCCA;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P68: a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising as operably linked components in 5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) a partial or complete coding sequence of PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P69: the rAAV according to embodiment P67 or embodiment P68, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10 or hu 37.

Embodiment P70: the rAAV according to embodiment P69, wherein the AAV capsid is from AAV 9.

Embodiment P71: the rAAV according to embodiment P69, wherein the AAV capsid is from AAV 8.

Embodiment P72: the rAAV according to embodiment P67 or embodiment P68, wherein the AAV capsid is an AAV9 variant capsid.

Embodiment P73: the rAAV according to any one of embodiments P67-P72, wherein the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a cytomegalovirus immediate early gene (CMV) promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, and a CAG promoter.

Embodiment P74: the rAAV according to embodiment P73, wherein the promoter is a CBA promoter.

Embodiment P75: the rAAV according to embodiment P67, wherein part or all of the coding sequence of the PCCA is a wild-type coding sequence.

Embodiment P76: the rAAV according to embodiment P75, wherein the coding sequence for PCCA comprises SEQ ID NO: 1.

embodiment P77: the rAAV according to embodiment P67, wherein the partial or complete coding sequence of PCCA is a codon optimized coding sequence.

Embodiment P78: the rAAV according to embodiment P77, wherein the coding sequence for PCCA comprises a sequence selected from SEQ ID NOs: 2-6.

Embodiment P79: the rAAV according to embodiment P68, wherein part or all of the coding sequence of the PCCB is a wild-type coding sequence.

Embodiment P80: the rAAV according to embodiment P79, wherein the coding sequence of PCCB comprises SEQ ID NO: 7.

embodiment P81: the rAAV according to embodiment P68, wherein part or the entire coding sequence of the PCCB is a codon optimized coding sequence.

Embodiment P82: the rAAV according to embodiment P81, wherein the coding sequence of the PCCB comprises a sequence selected from SEQ ID NOs: 8-12.

Embodiment P83: the rAAV according to any one of embodiments P67 to P82, wherein the enhancer is selected from the cytomegalovirus immediate early gene (CMV) enhancer, the transthyretin enhancer (enTTR), the chicken β -actin (CBA) enhancer, the En34 enhancer, and the ApoE enhancer.

Embodiment P84: the rAAV according to embodiment P83, wherein the enhancer is a CMV enhancer.

Embodiment P85: the rAAV according to any one of embodiments P67-P84, wherein the intron is selected from the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, or the hFIX intron.

Embodiment P86: the rAAV according to embodiment P85, wherein the intron is the SV40 small T intron.

Embodiment P87: the rAAV according to embodiment P85, wherein the intron is a rHBB intron.

Embodiment P88: the rAAV according to any one of embodiments P67 to P87, wherein the polyadenylation signal sequence is selected from a Bovine Growth Hormone (BGH) polyadenylation signal sequence, an SV40 polyadenylation signal sequence, and a rabbit β globin polyadenylation signal sequence.

Embodiment P89: the rAAV according to embodiment P88, wherein the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence.

Embodiment P90: the rAAV according to embodiment P88, wherein the polyadenylation signal sequence is an SV40 polyadenylation signal sequence.

Embodiment P91: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P92: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P93: the rAAV according to embodiment P91 or P92, wherein the AAV capsid is from AAV 9.

Embodiment P94: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P95: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' ITR sequence.

Embodiment P96: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) AAV 25' ITR sequences;

(b) a CMV enhancer sequence;

(c) a CBA promoter sequence;

(d) rHBB or SV40 small T intron sequences;

(e) selected from the group consisting of SEQ ID NOs: 1-6, the coding sequence of PCCA;

(f) a BGH or SV40 polyadenylation signal sequence; and

(g) AAV 23' ITR sequences.

Embodiment P97: a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) AAV 25' ITR sequences;

(b) a CMV enhancer sequence;

(c) a CBA promoter sequence;

(d) rHBB or SV40 small T intron sequences;

(e) selected from the group consisting of SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a BGH or SV40 polyadenylation signal sequence; and

(g) AAV 23' ITR sequences.

Embodiment P98: the rAAV according to any one of embodiments P67 to P97, wherein the vector genome comprises a consensus Kozak sequence located between vector genome elements (d) and (e).

Embodiment P99: the rAAV according to embodiment P98, wherein the consensus Kozak sequence comprises SEQ ID NO: 24.

embodiment P100: a composition comprising the rAAV of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

Embodiment P101: a method of treating Propionemia (PA) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of the rAAV of any one of embodiments P1-P99 or the composition of embodiment P100.

Embodiment P102: a method of treating Propionemia (PA) in a human subject, the method comprising administering to the human subject

(1) A therapeutically effective amount of a composition comprising a rAAV of any one of embodiments P1 to P33; and

(2) a therapeutically effective amount of a composition comprising a rAAV of any one of embodiments P34 to P66.

Embodiment P103: the method of embodiment P102, wherein the compositions of (1) and (2) are administered simultaneously.

Embodiment P104: the method of embodiment P102, wherein the compositions of (1) and (2) are administered sequentially.

Embodiment P105: the method of embodiment P102, wherein the compositions of (1) and (2) are administered separately.

Embodiment P106: a method of treating Propionemia (PA) in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering to the human subject a therapeutically effective amount of a composition comprising a rAAV of any one of embodiments P1-P33.

Embodiment P107: the method of embodiment P106, wherein the mutation in PCCA is selected from table 1.

Embodiment P108: a method of treating Propionemia (PA) in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering to the human subject a therapeutically effective amount of a composition comprising a rAAV of any one of embodiments P34-P66.

Embodiment P109: the method of embodiment P108, wherein the mutation in the PCCB is selected from table 2.

Embodiment P110: the method of any one of embodiments P101 to P109, wherein the recombinant virus, rAAV or composition is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, or intravenously.

Embodiment P111: the method of embodiment P110, wherein the rAAV or composition is administered intravenously.

Embodiment P112: the method of any one of embodiments P101 to P111, wherein the rAAV is at about 1x10¹¹To about 1x10¹⁴Genomic Copy (GC)/kg.

Embodiment P113: the method of embodiment P112, wherein the rAAV is at about 1x10¹²To about 1x10¹³Genomic Copy (GC)/kg.

Embodiment P114: the method according to any one of embodiments P110 to P113, wherein administering the rAAV comprises administration of a single dose of rAAV.

Embodiment P115: the method according to any one of embodiments P110 to P113, wherein administering the rAAV comprises administration of multiple doses of rAAV.

Embodiment P116: a recombinant nucleic acid encoding the polypeptide of SEQ ID NO: 16, wherein the nucleic acid sequence is identical to SEQ ID NO: 1 is less than 80% identical to the wild-type coding sequence.

Embodiment P117: the recombinant nucleic acid of embodiment P116, wherein the nucleic acid sequence comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 2-6, having at least 80% identity.

Embodiment P118: the recombinant nucleic acid of embodiment P117, wherein the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2-6.

Embodiment P119: the recombinant nucleic acid of any one of embodiments P116 to P118, wherein the nucleic acid sequence further comprises at the 3' terminus one or more stop codons selected from the group consisting of TGA, TAA, and TAG.

Embodiment P120: a recombinant nucleic acid encoding the polypeptide of SEQ ID NO: 17, wherein the nucleic acid sequence is identical to SEQ ID NO: 7 is less than 80% identical to the wild type coding sequence.

Embodiment P121: the recombinant nucleic acid of embodiment P120, wherein said nucleic acid sequence comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 8-12 sequences having at least 80% identity.

Embodiment P122: the recombinant nucleic acid of embodiment P121, wherein the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 8-12.

Embodiment P123: the recombinant nucleic acid of any one of embodiments P120 to P122, wherein the nucleic acid sequence further comprises at the 3' terminus one or more stop codons selected from the group consisting of TGA, TAA, and TAG.

Embodiment P124: a recombinant nucleic acid comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 80% identical.

Embodiment P125: the recombinant nucleic acid of embodiment P124, wherein the recombinant nucleic acid comprises a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 90% identical.

Embodiment P126: the recombinant nucleic acid of embodiment P125, wherein said recombinant nucleic acid comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 95% identical.

Embodiment P127: a recombinant nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 2-6.

Embodiment P128: the recombinant nucleic acid of any one of embodiments P124 to P127, wherein the nucleic acid sequence further comprises at the 3' terminus one or more stop codons selected from the group consisting of TGA, TAA, and TAG.

Embodiment P129: a recombinant nucleic acid comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12, which is at least 80% identical.

Embodiment P130: the recombinant nucleic acid of embodiment P129, wherein said recombinant nucleic acid comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 7-12, which is at least 90% identical.

Embodiment P131: the recombinant nucleic acid of embodiment P130, wherein said recombinant nucleic acid comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 7-12, which is at least 95% identical.

Embodiment P132: a recombinant nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 7-12.

Embodiment P133: the recombinant nucleic acid of any one of embodiments P129 to P132, wherein the nucleic acid sequence further comprises at the 3' terminus one or more stop codons selected from the group consisting of TGA, TAA, and TAG.

Embodiment P134: a rAAV comprising the recombinant nucleic acid according to any one of embodiments P116-P133.

Embodiment P135: a host cell comprising a recombinant nucleic acid according to any one of embodiments P116 to P133 or a rAAV of embodiment P134.

Embodiment P136: the host cell of embodiment P135, wherein the host cell is selected from the group consisting of HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19, and MRC-5 cells.

Is incorporated by reference

The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

Equality of nature

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Various structural elements of the different embodiments and various method steps disclosed may be used in various different combinations and permutations, and all such variations should be considered forms of the present disclosure. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Sequence listing

<110> Oltjirnix pharmaceuticals

<120> Gene therapy for treating propionic acidemia

<130> AJ4309PT2103

<150> 62/739,471

<151> 2018-10-01

<160> 39

<170> PatentIn version 3.5

<210> 1

<211> 2184

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

atggcggggt tctgggtcgg gacagcaccg ctggtcgctg ccggacggcg tgggcggtgg 60

ccgccgcagc agctgatgct gagcgcggcg ctgcggaccc tgaagcatgt tctgtactat 120

tcaagacagt gcttaatggt gtcccgtaat cttggttcag tgggatatga tcctaatgaa 180

aaaacttttg ataaaattct tgttgctaat agaggagaaa ttgcatgtcg ggttattaga 240

acttgcaaga agatgggcat taagacagtt gccatccaca gtgatgttga tgctagttct 300

gttcatgtga aaatggcgga tgaggctgtc tgtgttggcc cagctcccac cagtaaaagc 360

tacctcaaca tggatgccat catggaagcc attaagaaaa ccagggccca agctgtacat 420

ccaggttatg gattcctttc agaaaacaaa gaatttgcca gatgtttggc agcagaagat 480

gtcgttttca ttggacctga cacacatgct attcaagcca tgggcgacaa gattgaaagc 540

aaattattag ctaagaaagc agaggttaat acaatccctg gctttgatgg agtagtcaag 600

gatgcagaag aagctgtcag aattgcaagg gaaattggct accctgtcat gatcaaggcc 660

tcagcaggtg gtggtgggaa aggcatgcgc attgcttggg atgatgaaga gaccagggat 720

ggttttagat tgtcatctca agaagctgct tctagttttg gcgatgatag actactaata 780

gaaaaattta ttgataatcc tcgtcatata gaaatccagg ttctaggtga taaacatggg 840

aatgctttat ggcttaatga aagagagtgc tcaattcaga gaagaaatca gaaggtggtg 900

gaggaagcac caagcatttt tttggatgcg gagactcgaa gagcgatggg agaacaagct 960

gtagctcttg ccagagcagt aaaatattcc tctgctggga ccgtggagtt ccttgtggac 1020

tctaagaaga atttttattt cttggaaatg aatacaagac tccaggttga gcatcctgtc 1080

acagaatgca ttactggcct ggacctagtc caggaaatga tccgtgttgc taagggctac 1140

cctctcaggc acaaacaagc tgatattcgc atcaacggct gggcagttga atgtcgggtt 1200

tatgctgagg acccctacaa gtcttttggt ttaccatcta ttgggagatt gtctcagtac 1260

caagaaccgt tacatctacc tggtgtccga gtggacagtg gcatccaacc aggaagtgat 1320

attagcattt attatgatcc tatgatttca aaactaatca catatggctc tgatagaact 1380

gaggcactga agagaatggc agatgcactg gataactatg ttattcgagg tgttacacat 1440

aatattgcat tacttcgaga ggtgataatc aactcacgct ttgtaaaagg agacatcagc 1500

actaaatttc tctccgatgt gtatcctgat ggcttcaaag gacacatgct aaccaagagt 1560

gagaagaacc agttattggc aatagcatca tcattgtttg tggcattcca gttaagagca 1620

caacattttc aagaaaattc aagaatgcct gttattaaac cagacatagc caactgggag 1680

ctctcagtaa aattgcatga taaagttcat accgtagtag catcaaacaa tgggtcagtg 1740

ttctcggtgg aagttgatgg gtcgaaacta aatgtgacca gcacgtggaa cctggcttcg 1800

cccttattgt ctgtcagcgt tgatggcact cagaggactg tccagtgtct ttctcgagaa 1860

gcaggtggaa acatgagcat tcagtttctt ggtacagtgt acaaggtgaa tatcttaacc 1920

agacttgccg cagaattgaa caaatttatg ctggaaaaag tgactgagga cacaagcagt 1980

gttctgcgtt ccccgatgcc cggagtggtg gtggccgtct ctgtcaagcc tggagacgcg 2040

gtagcagaag gtcaagaaat ttgtgtgatt gaagccatga aaatgcagaa tagtatgaca 2100

gctgggaaaa ctggcacggt gaaatctgtg cactgtcaag ctggagacac agttggagaa 2160

ggggatctgc tcgtggagct ggaa 2184

<210> 2

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCA coding sequence-

Codon optimized

<400> 2

atggctggat tttgggtcgg aacggcccct ctcgtggccg ccggccgccg gggtcggtgg 60

ccgccgcaac agctgatgtt gtcggccgcg ctgcgcaccc ttaagcatgt gctgtactac 120

tcccggcaat gccttatggt gtccagaaac ctgggtagcg tgggctatga cccgaacgag 180

aaaaccttcg acaagattct ggtggccaac cggggggaaa ttgcctgccg ggtcatcagg 240

acttgcaaga agatgggcat caagaccgtc gccattcact ccgacgtgga cgcctcctcc 300

gtgcacgtga agatggcaga tgaagccgtc tgcgtgggcc ccgccccgac ctccaagtcc 360

taccttaaca tggacgcgat catggaagcc atcaaaaaga ccagagccca ggcagtgcac 420

ccgggatacg gctttctctc cgaaaacaag gagttcgcgc ggtgcctggc cgctgaagat 480

gtcgtgttca tcggccctga tacccacgcg atccaggcta tgggagacaa gatcgaatcc 540

aagctgctcg ccaagaaagc cgaagtcaac accatacctg ggtttgacgg cgtggtcaag 600

gacgcagaag aagccgtcag gattgcccgc gagatcggat accccgtgat gatcaaggca 660

tccgccgggg ggggaggaaa gggaatgcgc atcgcctggg atgacgaaga aacccgggac 720

ggcttcagac tctcgtcaca agaggccgcg tcctcattcg gggatgaccg gctcctgatt 780

gagaagttca ttgacaatcc tcggcacatc gagattcagg tcctgggcga taagcatgga 840

aacgccctgt ggctgaacga acgcgaatgc agcatccaga ggcggaacca gaaagtggtg 900

gaagaggccc catccatctt tctcgacgcc gagactcgga gagcgatggg tgaacaggcc 960

gtggccctgg cccgagccgt gaagtactcc agcgcgggga ctgtcgagtt cctggtggac 1020

agcaagaaga atttctactt cctggagatg aatactcggc tccaagtgga acaccccgtg 1080

accgaatgca ttaccggtct ggacctcgtc caagaaatga tccgcgtcgc caagggctac 1140

ccattgagac acaaacaggc cgacattcgg atcaacggat gggccgtcga gtgtcgcgtg 1200

tacgcggaag atccgtataa gtcgttcgga ctgccgtcca ttggtagact ctcgcagtac 1260

caagagccac tgcacctccc cggagtgcgc gtggactcag gcatccagcc cggaagcgac 1320

atctctatct actacgaccc catgatttcc aagttgatca cctacgggtc cgataggacc 1380

gaggcactga agcgcatggc tgacgcactt gacaactacg tgatccgcgg ggtcactcac 1440

aacattgccc tgctccgcga agtgatcatc aactcgcgct tcgtgaaggg cgacatctcc 1500

actaagttcc tgtccgacgt gtaccctgac ggtttcaagg gccatatgct gaccaagtcc 1560

gagaagaacc agctcctggc tatcgcctcc tccctgtttg tggcgttcca gctgagggcg 1620

cagcacttcc aggagaacag ccggatgccc gtgatcaagc ctgacatcgc caattgggag 1680

ctgtccgtga agctgcacga taaggtccat accgtggtgg catccaacaa cggatcggtg 1740

ttcagcgtgg aagtggacgg gtccaagctg aacgtgacca gcacatggaa cctggcgtcc 1800

cccctgttgt ctgtgtcggt cgatggcacg cagcgcactg tgcagtgcct ctcccgggaa 1860

gctggcggaa acatgagcat ccagttcctg ggtactgtgt acaaggtcaa cattctgact 1920

cggctggccg ccgagctgaa caagttcatg ttggaaaaag tcaccgaaga tacatcgtca 1980

gtcctgcgga gcccaatgcc tggagtcgtg gtggcggtgt cagtgaagcc cggcgatgct 2040

gtggccgaag gccaagagat ctgcgtgatc gaggccatga agatgcagaa ctcgatgacc 2100

gccggaaaga ccggtaccgt gaagtccgtg cattgtcaag cgggcgacac tgtgggagag 2160

ggagatctgc tcgtggagct ggag 2184

<210> 3

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCA coding sequence-

Codon optimized

<400> 3

atggccggct tctgggtcgg caccgcccct ctggtcgcgg ccggacgacg cggacgctgg 60

ccaccccagc aactgatgct gagcgcggcc ttgaggactc tgaagcacgt gctctactac 120

tcgcggcagt gcctgatggt gtcccggaat ctggggtccg tgggatacga ccctaacgaa 180

aagaccttcg ataagatcct cgtggcaaat cggggagaga tcgcgtgtcg cgtgatccgc 240

acgtgcaaga agatggggat caagactgtg gcaatccata gcgatgtgga tgcatcctcg 300

gtccacgtga agatggccga cgaagctgtg tgcgtgggac cggcgccgac ttcgaaatcg 360

tacctgaaca tggacgctat tatggaggcg atcaagaaaa cgcgcgccca agcggtccat 420

cccggttacg gattcctgag cgagaacaag gaatttgcac ggtgcctcgc tgccgaggac 480

gtggtgttta tcggtcccga cacccacgcc atccaagcta tgggggacaa gattgagtcc 540

aagctcctgg cgaaaaaggc agaggtcaac acaattcctg gtttcgacgg cgtcgtgaag 600

gacgccgaag aagccgtgcg catcgcgagg gaaatcggtt accctgtgat gattaaggcc 660

tccgccggcg gcggtggaaa gggaatgaga attgcctggg acgatgaaga aacccgcgac 720

ggattccgcc tgtcgagcca ggaagccgcc tcttccttcg gcgatgacag actgctgatc 780

gaaaagttca tcgataaccc cagacacatt gagatccaag tgctcgggga taagcacggc 840

aacgcccttt ggctgaacga gagagagtgc tccattcaac gccgcaatca gaaggtcgtg 900

gaggaagccc cgtcgatatt cctggatgcc gaaacccggc gggccatggg agagcaggct 960

gtcgcgttgg cgcgggccgt caagtacagc tcggccggga ccgtggaatt tctggtcgat 1020

tccaagaaga acttctattt cctggagatg aacaccagac tccaggtcga gcacccggtc 1080

actgagtgta tcaccgggct cgatctggtg caagagatga ttcgggtggc gaagggatat 1140

ccccttcggc ataaacaagc cgacatcagg atcaacggtt gggccgtgga atgcagggtc 1200

tacgccgagg acccctacaa gagcttcggc ctgcccagca tcggccgcct gtcacagtat 1260

caggaaccgc tgcatcttcc gggcgtgcgg gtcgacagcg gaattcagcc tggctcagat 1320

atctccatct actacgatcc aatgatctca aagctgatta cttatggatc cgaccggacc 1380

gaagccctta agcgaatggc cgacgccctg gacaactacg tgatccgggg agtgacccac 1440

aacatcgcct tgctgcggga agtgatcatt aacagcagat tcgtgaaggg agacatcagc 1500

accaagttcc tgtcggatgt ctacccggac gggttcaaag ggcacatgct tactaagtcc 1560

gagaagaatc agctgctcgc cattgcgtca agcttgttcg tggcctttca actccgggcc 1620

cagcacttcc aggaaaactc ccgcatgcca gtcattaagc cggacatcgc caactgggaa 1680

ctcagcgtga agctccatga caaagtgcat accgtggtgg ccagcaacaa cggtagcgtg 1740

ttctcagtcg aggtcgatgg ctcgaagctc aacgtcactt ccacttggaa cttggccagc 1800

ccgctgctgt ccgtgtccgt ggacggaacc cagaggaccg tgcagtgtct gtcgagagaa 1860

gccggcggca acatgtcaat ccagttcctg ggaaccgtgt acaaggtcaa catcctgacc 1920

agactggccg ccgaactgaa caagtttatg ctcgagaaag tgaccgagga cactagctcc 1980

gtgctgcgct cccctatgcc cggagtggtc gtggcagtgt ccgtgaagcc gggcgacgcc 2040

gtggccgagg gacaggaaat ctgtgtgatc gaagcgatga agatgcagaa ttcaatgacc 2100

gcgggaaaga ctgggaccgt gaagtctgtg cactgccagg ctggcgatac cgtgggggag 2160

ggcgaccttc tggtggaact cgag 2184

<210> 4

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCA coding sequence-

Codon optimized

<400> 4

atggctgggt tttgggtggg gacagctcct ctggtggctg ctgggaggag ggggaggtgg 60

cctcctcagc agctgatgct gtctgctgct ctgaggacac tgaagcatgt gctgtattat 120

tctaggcagt gtctgatggt gtctaggaat ctggggtctg tggggtatga tcctaatgag 180

aagacatttg ataagattct ggtggctaat aggggggaga ttgcttgtag ggtgattagg 240

acatgtaaga agatggggat taagacagtg gctattcatt ctgatgtgga tgcttcttct 300

gtgcatgtga agatggctga tgaggctgtg tgtgtggggc ctgctcctac atctaagtct 360

tatctgaata tggatgctat tatggaggct attaagaaga caagggctca ggctgtgcat 420

cctgggtatg ggtttctgtc tgagaataag gagtttgcta ggtgtctggc tgctgaggat 480

gtggtgttta ttgggcctga tacacatgct attcaggcta tgggggataa gattgagtct 540

aagctgctgg ctaagaaggc tgaggtgaat acaattcctg ggtttgatgg ggtggtgaag 600

gatgctgagg aggctgtgag gattgctagg gagattgggt atcctgtgat gattaaggct 660

tctgctgggg ggggggggaa ggggatgagg attgcttggg atgatgagga gacaagggat 720

gggtttaggc tgtcttctca ggaggctgct tcttcttttg gggatgatag gctgctgatt 780

gagaagttta ttgataatcc taggcatatt gagattcagg tgctggggga taagcatggg 840

aatgctctgt ggctgaatga gagggagtgt tctattcaga ggaggaatca gaaggtggtg 900

gaggaggctc cttctatttt tctggatgct gagacaagga gggctatggg ggagcaggct 960

gtggctctgg ctagggctgt gaagtattct tctgctggga cagtggagtt tctggtggat 1020

tctaagaaga atttttattt tctggagatg aatacaaggc tgcaggtgga gcatcctgtg 1080

acagagtgta ttacagggct ggatctggtg caggagatga ttagggtggc taaggggtat 1140

cctctgaggc ataagcaggc tgatattagg attaatgggt gggctgtgga gtgtagggtg 1200

tatgctgagg atccttataa gtcttttggg ctgccttcta ttgggaggct gtctcagtat 1260

caggagcctc tgcatctgcc tggggtgagg gtggattctg ggattcagcc tgggtctgat 1320

atttctattt attatgatcc tatgatttct aagctgatta catatgggtc tgataggaca 1380

gaggctctga agaggatggc tgatgctctg gataattatg tgattagggg ggtgacacat 1440

aatattgctc tgctgaggga ggtgattatt aattctaggt ttgtgaaggg ggatatttct 1500

acaaagtttc tgtctgatgt gtatcctgat gggtttaagg ggcatatgct gacaaagtct 1560

gagaagaatc agctgctggc tattgcttct tctctgtttg tggcttttca gctgagggct 1620

cagcattttc aggagaattc taggatgcct gtgattaagc ctgatattgc taattgggag 1680

ctgtctgtga agctgcatga taaggtgcat acagtggtgg cttctaataa tgggtctgtg 1740

ttttctgtgg aggtggatgg gtctaagctg aatgtgacat ctacatggaa tctggcttct 1800

cctctgctgt ctgtgtctgt ggatgggaca cagaggacag tgcagtgtct gtctagggag 1860

gctgggggga atatgtctat tcagtttctg gggacagtgt ataaggtgaa tattctgaca 1920

aggctggctg ctgagctgaa taagtttatg ctggagaagg tgacagagga tacatcttct 1980

gtgctgaggt ctcctatgcc tggggtggtg gtggctgtgt ctgtgaagcc tggggatgct 2040

gtggctgagg ggcaggagat ttgtgtgatt gaggctatga agatgcagaa ttctatgaca 2100

gctgggaaga cagggacagt gaagtctgtg cattgtcagg ctggggatac agtgggggag 2160

ggggatctgc tggtggagct ggag 2184

<210> 5

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCA coding sequence-

Codon optimized

<400> 5

atggcagggt tttgggtggg gacagcacca ctggtggcag cagggaggag ggggaggtgg 60

ccaccacagc agctgatgct gtcagcagca ctgaggacac tgaagcatgt gctgtattat 120

tcaaggcagt gtctgatggt gtcaaggaat ctggggtcag tggggtatga tccaaatgag 180

aagacatttg ataagattct ggtggcaaat aggggggaga ttgcatgtag ggtgattagg 240

acatgtaaga agatggggat taagacagtg gcaattcatt cagatgtgga tgcatcatca 300

gtgcatgtga agatggcaga tgaggcagtg tgtgtggggc cagcaccaac atcaaagtca 360

tatctgaata tggatgcaat tatggaggca attaagaaga caagggcaca ggcagtgcat 420

ccagggtatg ggtttctgtc agagaataag gagtttgcaa ggtgtctggc agcagaggat 480

gtggtgttta ttgggccaga tacacatgca attcaggcaa tgggggataa gattgagtca 540

aagctgctgg caaagaaggc agaggtgaat acaattccag ggtttgatgg ggtggtgaag 600

gatgcagagg aggcagtgag gattgcaagg gagattgggt atccagtgat gattaaggca 660

tcagcagggg ggggggggaa ggggatgagg attgcatggg atgatgagga gacaagggat 720

gggtttaggc tgtcatcaca ggaggcagca tcatcatttg gggatgatag gctgctgatt 780

gagaagttta ttgataatcc aaggcatatt gagattcagg tgctggggga taagcatggg 840

aatgcactgt ggctgaatga gagggagtgt tcaattcaga ggaggaatca gaaggtggtg 900

gaggaggcac catcaatttt tctggatgca gagacaagga gggcaatggg ggagcaggca 960

gtggcactgg caagggcagt gaagtattca tcagcaggga cagtggagtt tctggtggat 1020

tcaaagaaga atttttattt tctggagatg aatacaaggc tgcaggtgga gcatccagtg 1080

acagagtgta ttacagggct ggatctggtg caggagatga ttagggtggc aaaggggtat 1140

ccactgaggc ataagcaggc agatattagg attaatgggt gggcagtgga gtgtagggtg 1200

tatgcagagg atccatataa gtcatttggg ctgccatcaa ttgggaggct gtcacagtat 1260

caggagccac tgcatctgcc aggggtgagg gtggattcag ggattcagcc agggtcagat 1320

atttcaattt attatgatcc aatgatttca aagctgatta catatgggtc agataggaca 1380

gaggcactga agaggatggc agatgcactg gataattatg tgattagggg ggtgacacat 1440

aatattgcac tgctgaggga ggtgattatt aattcaaggt ttgtgaaggg ggatatttca 1500

acaaagtttc tgtcagatgt gtatccagat gggtttaagg ggcatatgct gacaaagtca 1560

gagaagaatc agctgctggc aattgcatca tcactgtttg tggcatttca gctgagggca 1620

cagcattttc aggagaattc aaggatgcca gtgattaagc cagatattgc aaattgggag 1680

ctgtcagtga agctgcatga taaggtgcat acagtggtgg catcaaataa tgggtcagtg 1740

ttttcagtgg aggtggatgg gtcaaagctg aatgtgacat caacatggaa tctggcatca 1800

ccactgctgt cagtgtcagt ggatgggaca cagaggacag tgcagtgtct gtcaagggag 1860

gcagggggga atatgtcaat tcagtttctg gggacagtgt ataaggtgaa tattctgaca 1920

aggctggcag cagagctgaa taagtttatg ctggagaagg tgacagagga tacatcatca 1980

gtgctgaggt caccaatgcc aggggtggtg gtggcagtgt cagtgaagcc aggggatgca 2040

gtggcagagg ggcaggagat ttgtgtgatt gaggcaatga agatgcagaa ttcaatgaca 2100

gcagggaaga cagggacagt gaagtcagtg cattgtcagg caggggatac agtgggggag 2160

ggggatctgc tggtggagct ggag 2184

<210> 6

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCA coding sequence-

Codon optimized

<400> 6

atggctggtt tttgggtagg tacagcacca ctagtagcag caggtaggag gggtaggtgg 60

ccaccacaac aactaatgct atcagcagca ctaaggacac taaagcatgt actatattat 120

tcaaggcaat gtctaatggt atcaaggaat ctggggtctg tggggtatga tcctaatgag 180

aagacatttg ataagattct ggtggctaat aggggggaga ttgcttgtag ggtgattagg 240

acatgtaaga agatggggat taagacagtg gctattcatt ctgatgtgga tgcttcttct 300

gtgcatgtga agatggctga tgaggctgtg tgtgtggggc ctgctcctac atctaagtct 360

tatctgaata tggatgctat tatggaggct attaagaaga caagggctca ggctgtgcat 420

cctgggtatg ggtttctgtc tgagaataag gagtttgcta ggtgtctggc tgctgaggat 480

gtggtgttta ttgggcctga tacacatgct attcaggcta tgggggataa gattgagtct 540

aagctgctgg ctaagaaggc tgaggtgaat acaattcctg ggtttgatgg ggtggtgaag 600

gatgctgagg aggctgtgag gattgctagg gagattgggt atcctgtgat gattaaggct 660

tctgctgggg ggggggggaa ggggatgagg attgcttggg atgatgagga gacaagggat 720

gggtttaggc tgtcttctca ggaggctgct tcttcttttg gggatgatag gctgctgatt 780

gagaagttta ttgataatcc taggcatatt gagattcagg tgctggggga taagcatggg 840

aatgctctgt ggctgaatga gagggagtgt tctattcaga ggaggaatca gaaggtggtg 900

gaggaggctc cttctatttt tctggatgct gagacaagga gggctatggg ggagcaggct 960

gtggctctgg ctagggctgt gaagtattct tctgctggga cagtggagtt tctggtggat 1020

tctaagaaga atttttattt tctggagatg aatacaaggc tgcaggtgga gcatcctgtg 1080

acagagtgta ttacagggct ggatctggtg caggagatga ttagggtggc taaggggtat 1140

cctctgaggc ataagcaggc tgatattagg attaatgggt gggctgtgga gtgtagggtg 1200

tatgctgagg atccttataa gtcttttggg ctgccttcta ttgggaggct gtctcagtat 1260

caggagcctc tgcatctgcc tggggtgagg gtggattctg ggattcagcc tgggtctgat 1320

atttctattt attatgatcc tatgatttct aagctgatta catatgggtc tgataggaca 1380

gaggctctga agaggatggc tgatgctctg gataattatg tgattagggg ggtgacacat 1440

aatattgctc tgctgaggga ggtgattatt aattctaggt ttgtgaaggg ggatatttct 1500

acaaagtttc tgtctgatgt gtatcctgat gggtttaagg ggcatatgct gacaaagtct 1560

gagaagaatc agctgctggc tattgcttct tctctgtttg tggcttttca gctgagggct 1620

cagcattttc aggagaattc taggatgcct gtgattaagc ctgatattgc taattgggag 1680

ctgtctgtga agctgcatga taaggtgcat acagtggtgg cttctaataa tgggtctgtg 1740

ttttctgtgg aggtggatgg gtctaagctg aatgtgacat ctacatggaa tctggcttct 1800

cctctgctgt ctgtgtctgt ggatgggaca cagaggacag tgcagtgtct gtctagggag 1860

gctgggggga atatgtctat tcagtttctg gggacagtgt ataaggtgaa tattctgaca 1920

aggctggctg ctgagctgaa taagtttatg ctggagaagg tgacagagga tacatcttct 1980

gtgctgaggt ctcctatgcc tggggtggtg gtggctgtgt ctgtgaagcc tggggatgct 2040

gtggctgagg ggcaggagat ttgtgtgatt gaggctatga agatgcagaa ttctatgaca 2100

gctgggaaga cagggacagt gaagtctgtg cattgtcagg ctggggatac agtgggggag 2160

ggggatctgc tggtggagct ggag 2184

<210> 7

<211> 1617

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 7

atggcggcgg cattacgggt ggcggcggtc ggggcaaggc tcagcgttct ggcgagcggt 60

ctccgcgccg cggtccgcag cctttgcagc caggccacct ctgttaacga acgcatcgaa 120

aacaagcgcc ggaccgcgct gctgggaggg ggccaacgcc gtattgacgc gcagcacaag 180

cgaggaaagc taacagccag ggagaggatc agtctcttgc tggaccctgg cagctttgtt 240

gagagcgaca tgtttgtgga acacagatgt gcagattttg gaatggctgc tgataagaat 300

aagtttcctg gagacagcgt ggtcactgga cgaggccgaa tcaatggaag attggtttat 360

gtcttcagtc aggattttac agtttttgga ggcagtctgt caggagcaca tgcccaaaag 420

atctgcaaaa tcatggacca ggccataacg gtgggggctc cagtgattgg gctgaatgac 480

tctgggggag cacggatcca agaaggagtg gagtctttgg ctggctatgc agacatcttt 540

ctgaggaatg ttacggcatc cggagtcatc cctcagattt ctctgatcat gggcccatgt 600

gctggtgggg ccgtctactc cccagcccta acagacttca cgttcatggt aaaggacacc 660

tcctacctgt tcatcactgg ccctgatgtt gtgaagtctg tcaccaatga ggatgttacc 720

caggaggagc tcggtggtgc caagacccac accaccatgt caggtgtggc ccacagagct 780

tttgaaaatg atgttgatgc cttgtgtaat ctccgggatt tcttcaacta cctgcccctg 840

agcagtcagg acccggctcc cgtccgtgag tgccacgatc ccagtgaccg tctggttcct 900

gagcttgaca caattgtccc tttggaatca accaaagcct acaacatggt ggacatcata 960

cactctgttg ttgatgagcg tgaatttttt gagatcatgc ccaattatgc caagaacatc 1020

attgttggtt ttgcaagaat gaatgggagg actgttggaa ttgttggcaa ccaacctaag 1080

gtggcctcag gatgcttgga tattaattca tctgtgaaag gggctcgttt tgtcagattc 1140

tgtgatgcat tcaatattcc actcatcact tttgttgatg tccctggctt tctacctggc 1200

acagcacagg aatacggggg catcatccgg catggtgcca agcttctcta cgcatttgct 1260

gaggcaactg tacccaaagt cacagtcatc accaggaagg cctatggagg tgcctatgat 1320

gtcatgagct ctaagcacct ttgtggtgat accaactatg cctggcccac cgcagagatt 1380

gcagtcatgg gagcaaaggg cgctgtggag atcatcttca aagggcatga gaatgtggaa 1440

gctgctcagg cagagtacat cgagaagttt gccaaccctt tccctgcagc agtgcgaggg 1500

tttgtggatg acatcatcca accttcttcc acacgtgccc gaatctgctg tgacctggat 1560

gtcttggcca gcaagaaggt acaacgtcct tggagaaaac atgcaaatat tccattg 1617

<210> 8

<211> 1617

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCB coding sequence-

Codon optimized

<400> 8

atggctgccg ccctgcgcgt ggcggccgtg ggagcaagac tgtccgtgct ggcgtcgggc 60

ttgagagcgg ccgtgcggag cctgtgctca caagcaacct cggtgaacga acgcatcgag 120

aacaagcgca ggactgcgct gctgggcggg ggccagcgca ggatcgacgc acagcataag 180

cgcggaaagc tgaccgcccg cgagcggatt tccctgctcc tggatcctgg aagcttcgtg 240

gagtccgaca tgttcgtgga gcaccgctgc gccgacttcg ggatggctgc cgacaagaac 300

aagttccccg gggactcagt ggtcactggt cgcggaagaa tcaatggccg gctcgtctac 360

gtgttctcac aagactttac tgtgttcggc ggctccctgt cgggagccca cgcgcaaaag 420

atctgcaaga ttatggatca ggccatcact gtgggagcgc ctgtgattgg actcaacgac 480

tccgggggag caagaatcca ggaaggagtg gaaagccttg ccggctacgc tgacatcttc 540

ctccggaacg tgaccgcctc tggagtgatt ccgcaaatct ccctgatcat gggaccatgt 600

gccgggggcg ccgtgtactc cccggcgctg actgacttca ctttcatggt caaggacaca 660

tcctacctgt tcatcaccgg tcccgacgtc gtgaagtccg tgaccaacga ggatgtgacc 720

caggaagaac tggggggggc caagacgcat accaccatgt cgggagtggc ccaccgggcc 780

ttcgagaacg atgtggacgc cttgtgcaac cttcgggact tcttcaatta tctcccgctg 840

agcagccagg atccggcccc agtgcgggaa tgccacgacc cttcggatcg gttggtgcct 900

gagctggata ccatcgtgcc cctcgaatcc accaaggctt acaacatggt cgacatcatt 960

cactccgtgg tggacgagag ggaattcttc gagattatgc cgaactacgc caagaacatc 1020

attgtcggat tcgcccgcat gaacggtcga actgtgggca ttgtcggaaa ccagcctaaa 1080

gtggcctccg gttgcctgga catcaactca agcgtgaagg gtgccagatt tgtgcggttt 1140

tgtgacgcgt tcaatattcc gctgatcacc ttcgtcgacg tcccgggctt cctgcctggg 1200

accgcccagg aatacggcgg catcatcaga cacggcgcga agctcctcta cgcgttcgcg 1260

gaagccaccg tgcccaaggt caccgtgatc actcgcaagg catacggcgg cgcatacgat 1320

gtgatgtcct ccaagcacct gtgtggcgac accaactacg cctggcccac cgccgagatc 1380

gccgtgatgg gtgccaaggg tgctgtcgag atcatcttca agggacatga aaacgtggaa 1440

gctgcccagg ccgagtacat tgaaaagttc gctaacccct tccctgccgc cgtgcgggga 1500

tttgtggatg acattatcca gccgagctcg accagggcca gaatctgctg cgatcttgat 1560

gtgttggcca gcaaaaaggt ccagcggccc tggcggaaac acgccaacat tccactg 1617

<210> 9

<211> 1617

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCB coding sequence-

Codon optimized

<400> 9

atggccgcgg cgcttagagt ggccgctgtg ggagccaggc tgagcgtgct ggccagcggt 60

ctgcgcgccg cagtgcgctc gctgtgtagc caggctacct ccgtgaatga gcggatcgaa 120

aacaagcggc gcaccgccct gttgggcggc ggacagcggc gaattgacgc ccaacacaag 180

cggggaaagc tcactgcgag ggaaagaatc tcactgctgc tcgaccccgg gtcgttcgtg 240

gaatcggata tgtttgtcga acatagatgc gcagatttcg gaatggccgc tgacaagaac 300

aagttcccgg gagattccgt cgtgaccgga agggggcgca ttaacgggag acttgtgtac 360

gtgttcagcc aggatttcac ggtgttcggc ggatcactga gcggtgcaca tgcacagaag 420

atctgcaaga tcatggacca ggccattacc gtcggggcac ctgtgatcgg cctgaatgat 480

tcgggcggag cccggattca agagggcgtg gagtcactcg cgggttacgc cgacattttc 540

ctgcggaacg tcaccgcctc cggcgtgatc cctcaaatca gcctcattat gggcccctgc 600

gcgggcggtg ccgtctactc acccgctctg accgatttta ccttcatggt caaggacacc 660

tcctatctgt ttatcactgg accagatgtg gtcaagtccg tgaccaacga ggacgtcact 720

caggaagaac tcggtggagc aaagacccac actactatgt ccggggtcgc gcatagagct 780

ttcgaaaacg acgtcgatgc tctctgtaac ctgagggatt tcttcaacta ccttccactg 840

tcgtcgcaag acccagcccc cgtgcgcgag tgccacgatc cctccgaccg cctggtgccg 900

gaactcgaca ctattgtccc tctggagtca accaaggcct acaacatggt ggacatcatc 960

catagcgtcg tggatgaacg ggagttcttc gaaatcatgc ccaactatgc gaaaaatatc 1020

atcgtgggct ttgcgcggat gaacggccgc accgtgggca tagtgggcaa ccagccgaag 1080

gtcgcgtcgg gatgcctcga tatcaacagc tctgtgaagg gagcgcggtt cgtgcgcttc 1140

tgcgacgcct tcaacatccc cttgatcacc ttcgtggatg tgcctgggtt cttgcctgga 1200

accgcccagg aatacggggg gatcattcgg cacggagcaa aactgctgta cgccttcgcc 1260

gaggccactg tgccgaaagt gacagtgatt acccggaagg cctacggggg tgcctacgac 1320

gtgatgagct ccaagcacct gtgcggagac accaattacg cgtggcctac tgctgaaatt 1380

gctgtcatgg gagccaaggg cgccgtggaa atcattttca agggccacga aaacgtcgag 1440

gccgcccaag ctgagtacat cgagaagttt gccaacccgt ttcctgcggc tgtgcgcggc 1500

ttcgtcgacg atatcattca gccctcgtcc actcgcgccc gcatttgttg tgacctcgac 1560

gtgctggcgt ccaagaaagt gcaaagaccg tggagaaagc atgcaaacat cccgctc 1617

<210> 10

<211> 1617

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCB coding sequence-

Codon optimized

<400> 10

atggctgctg ctctgagggt ggctgctgtg ggggctaggc tgtctgtgct ggcttctggg 60

ctgagggctg ctgtgaggtc tctgtgttct caggctacat ctgtgaatga gaggattgag 120

aataagagga ggacagctct gctggggggg gggcagagga ggattgatgc tcagcataag 180

agggggaagc tgacagctag ggagaggatt tctctgctgc tggatcctgg gtcttttgtg 240

gagtctgata tgtttgtgga gcataggtgt gctgattttg ggatggctgc tgataagaat 300

aagtttcctg gggattctgt ggtgacaggg agggggagga ttaatgggag gctggtgtat 360

gtgttttctc aggattttac agtgtttggg gggtctctgt ctggggctca tgctcagaag 420

atttgtaaga ttatggatca ggctattaca gtgggggctc ctgtgattgg gctgaatgat 480

tctggggggg ctaggattca ggagggggtg gagtctctgg ctgggtatgc tgatattttt 540

ctgaggaatg tgacagcttc tggggtgatt cctcagattt ctctgattat ggggccttgt 600

gctggggggg ctgtgtattc tcctgctctg acagatttta catttatggt gaaggataca 660

tcttatctgt ttattacagg gcctgatgtg gtgaagtctg tgacaaatga ggatgtgaca 720

caggaggagc tggggggggc taagacacat acaacaatgt ctggggtggc tcatagggct 780

tttgagaatg atgtggatgc tctgtgtaat ctgagggatt tttttaatta tctgcctctg 840

tcttctcagg atcctgctcc tgtgagggag tgtcatgatc cttctgatag gctggtgcct 900

gagctggata caattgtgcc tctggagtct acaaaggctt ataatatggt ggatattatt 960

cattctgtgg tggatgagag ggagtttttt gagattatgc ctaattatgc taagaatatt 1020

attgtggggt ttgctaggat gaatgggagg acagtgggga ttgtggggaa tcagcctaag 1080

gtggcttctg ggtgtctgga tattaattct tctgtgaagg gggctaggtt tgtgaggttt 1140

tgtgatgctt ttaatattcc tctgattaca tttgtggatg tgcctgggtt tctgcctggg 1200

acagctcagg agtatggggg gattattagg catggggcta agctgctgta tgcttttgct 1260

gaggctacag tgcctaaggt gacagtgatt acaaggaagg cttatggggg ggcttatgat 1320

gtgatgtctt ctaagcatct gtgtggggat acaaattatg cttggcctac agctgagatt 1380

gctgtgatgg gggctaaggg ggctgtggag attattttta aggggcatga gaatgtggag 1440

gctgctcagg ctgagtatat tgagaagttt gctaatcctt ttcctgctgc tgtgaggggg 1500

tttgtggatg atattattca gccttcttct acaagggcta ggatttgttg tgatctggat 1560

gtgctggctt ctaagaaggt gcagaggcct tggaggaagc atgctaatat tcctctg 1617

<210> 11

<211> 1617

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCB coding sequence-

Codon optimized

<400> 11

atggcagcag cactgagggt ggcagcagtg ggggcaaggc tgtcagtgct ggcatcaggg 60

ctgagggcag cagtgaggtc actgtgttca caggcaacat cagtgaatga gaggattgag 120

aataagagga ggacagcact gctggggggg gggcagagga ggattgatgc acagcataag 180

agggggaagc tgacagcaag ggagaggatt tcactgctgc tggatccagg gtcatttgtg 240

gagtcagata tgtttgtgga gcataggtgt gcagattttg ggatggcagc agataagaat 300

aagtttccag gggattcagt ggtgacaggg agggggagga ttaatgggag gctggtgtat 360

gtgttttcac aggattttac agtgtttggg gggtcactgt caggggcaca tgcacagaag 420

atttgtaaga ttatggatca ggcaattaca gtgggggcac cagtgattgg gctgaatgat 480

tcaggggggg caaggattca ggagggggtg gagtcactgg cagggtatgc agatattttt 540

ctgaggaatg tgacagcatc aggggtgatt ccacagattt cactgattat ggggccatgt 600

gcaggggggg cagtgtattc accagcactg acagatttta catttatggt gaaggataca 660

tcatatctgt ttattacagg gccagatgtg gtgaagtcag tgacaaatga ggatgtgaca 720

caggaggagc tggggggggc aaagacacat acaacaatgt caggggtggc acatagggca 780

tttgagaatg atgtggatgc actgtgtaat ctgagggatt tttttaatta tctgccactg 840

tcatcacagg atccagcacc agtgagggag tgtcatgatc catcagatag gctggtgcca 900

gagctggata caattgtgcc actggagtca acaaaggcat ataatatggt ggatattatt 960

cattcagtgg tggatgagag ggagtttttt gagattatgc caaattatgc aaagaatatt 1020

attgtggggt ttgcaaggat gaatgggagg acagtgggga ttgtggggaa tcagccaaag 1080

gtggcatcag ggtgtctgga tattaattca tcagtgaagg gggcaaggtt tgtgaggttt 1140

tgtgatgcat ttaatattcc actgattaca tttgtggatg tgccagggtt tctgccaggg 1200

acagcacagg agtatggggg gattattagg catggggcaa agctgctgta tgcatttgca 1260

gaggcaacag tgccaaaggt gacagtgatt acaaggaagg catatggggg ggcatatgat 1320

gtgatgtcat caaagcatct gtgtggggat acaaattatg catggccaac agcagagatt 1380

gcagtgatgg gggcaaaggg ggcagtggag attattttta aggggcatga gaatgtggag 1440

gcagcacagg cagagtatat tgagaagttt gcaaatccat ttccagcagc agtgaggggg 1500

tttgtggatg atattattca gccatcatca acaagggcaa ggatttgttg tgatctggat 1560

gtgctggcat caaagaaggt gcagaggcca tggaggaagc atgcaaatat tccactg 1617

<210> 12

<211> 1617

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCCB coding sequence-

Codon optimized

<400> 12

atggctgcag cactaagggt agcagcagta ggtgcaaggc tatcagtact agcatcaggt 60

ctaagggcag cagtaaggtc actatgttca caagcaacat cagtaaatga aaggatagaa 120

aataagagga ggacagcact actaggtggt gggcagagga ggattgatgc tcagcataag 180

agggggaagc tgacagctag ggagaggatt tctctgctgc tggatcctgg gtcttttgtg 240

gagtctgata tgtttgtgga gcataggtgt gctgattttg ggatggctgc tgataagaat 300

aagtttcctg gggattctgt ggtgacaggg agggggagga ttaatgggag gctggtgtat 360

gtgttttctc aggattttac agtgtttggg gggtctctgt ctggggctca tgctcagaag 420

atttgtaaga ttatggatca ggctattaca gtgggggctc ctgtgattgg gctgaatgat 480

tctggggggg ctaggattca ggagggggtg gagtctctgg ctgggtatgc tgatattttt 540

ctgaggaatg tgacagcttc tggggtgatt cctcagattt ctctgattat ggggccttgt 600

gctggggggg ctgtgtattc tcctgctctg acagatttta catttatggt gaaggataca 660

tcttatctgt ttattacagg gcctgatgtg gtgaagtctg tgacaaatga ggatgtgaca 720

caggaggagc tggggggggc taagacacat acaacaatgt ctggggtggc tcatagggct 780

tttgagaatg atgtggatgc tctgtgtaat ctgagggatt tttttaatta tctgcctctg 840

tcttctcagg atcctgctcc tgtgagggag tgtcatgatc cttctgatag gctggtgcct 900

gagctggata caattgtgcc tctggagtct acaaaggctt ataatatggt ggatattatt 960

cattctgtgg tggatgagag ggagtttttt gagattatgc ctaattatgc taagaatatt 1020

attgtggggt ttgctaggat gaatgggagg acagtgggga ttgtggggaa tcagcctaag 1080

gtggcttctg ggtgtctgga tattaattct tctgtgaagg gggctaggtt tgtgaggttt 1140

tgtgatgctt ttaatattcc tctgattaca tttgtggatg tgcctgggtt tctgcctggg 1200

acagctcagg agtatggggg gattattagg catggggcta agctgctgta tgcttttgct 1260

gaggctacag tgcctaaggt gacagtgatt acaaggaagg cttatggggg ggcttatgat 1320

gtgatgtctt ctaagcatct gtgtggggat acaaattatg cttggcctac agctgagatt 1380

gctgtgatgg gggctaaggg ggctgtggag attattttta aggggcatga gaatgtggag 1440

gctgctcagg ctgagtatat tgagaagttt gctaatcctt ttcctgctgc tgtgaggggg 1500

tttgtggatg atattattca gccttcttct acaagggcta ggatttgttg tgatctggat 1560

gtgctggctt ctaagaaggt gcagaggcct tggaggaagc atgctaatat tcctctg 1617

<210> 13

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: AAV9 nucleic acid sequence

<400> 13

atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740

gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800

atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860

aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920

aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980

gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040

gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100

tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160

tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211

<210> 14

<211> 736

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: AAV9 amino acid sequence

<400> 14

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 15

<211> 145

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: AAV2 ITR

<400> 15

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcct 145

<210> 16

<211> 728

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 16

Met Ala Gly Phe Trp Val Gly Thr Ala Pro Leu Val Ala Ala Gly Arg

1 5 10 15

Arg Gly Arg Trp Pro Pro Gln Gln Leu Met Leu Ser Ala Ala Leu Arg

20 25 30

Thr Leu Lys His Val Leu Tyr Tyr Ser Arg Gln Cys Leu Met Val Ser

35 40 45

Arg Asn Leu Gly Ser Val Gly Tyr Asp Pro Asn Glu Lys Thr Phe Asp

50 55 60

Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Ala Cys Arg Val Ile Arg

65 70 75 80

Thr Cys Lys Lys Met Gly Ile Lys Thr Val Ala Ile His Ser Asp Val

85 90 95

Asp Ala Ser Ser Val His Val Lys Met Ala Asp Glu Ala Val Cys Val

100 105 110

Gly Pro Ala Pro Thr Ser Lys Ser Tyr Leu Asn Met Asp Ala Ile Met

115 120 125

Glu Ala Ile Lys Lys Thr Arg Ala Gln Ala Val His Pro Gly Tyr Gly

130 135 140

Phe Leu Ser Glu Asn Lys Glu Phe Ala Arg Cys Leu Ala Ala Glu Asp

145 150 155 160

Val Val Phe Ile Gly Pro Asp Thr His Ala Ile Gln Ala Met Gly Asp

165 170 175

Lys Ile Glu Ser Lys Leu Leu Ala Lys Lys Ala Glu Val Asn Thr Ile

180 185 190

Pro Gly Phe Asp Gly Val Val Lys Asp Ala Glu Glu Ala Val Arg Ile

195 200 205

Ala Arg Glu Ile Gly Tyr Pro Val Met Ile Lys Ala Ser Ala Gly Gly

210 215 220

Gly Gly Lys Gly Met Arg Ile Ala Trp Asp Asp Glu Glu Thr Arg Asp

225 230 235 240

Gly Phe Arg Leu Ser Ser Gln Glu Ala Ala Ser Ser Phe Gly Asp Asp

245 250 255

Arg Leu Leu Ile Glu Lys Phe Ile Asp Asn Pro Arg His Ile Glu Ile

260 265 270

Gln Val Leu Gly Asp Lys His Gly Asn Ala Leu Trp Leu Asn Glu Arg

275 280 285

Glu Cys Ser Ile Gln Arg Arg Asn Gln Lys Val Val Glu Glu Ala Pro

290 295 300

Ser Ile Phe Leu Asp Ala Glu Thr Arg Arg Ala Met Gly Glu Gln Ala

305 310 315 320

Val Ala Leu Ala Arg Ala Val Lys Tyr Ser Ser Ala Gly Thr Val Glu

325 330 335

Phe Leu Val Asp Ser Lys Lys Asn Phe Tyr Phe Leu Glu Met Asn Thr

340 345 350

Arg Leu Gln Val Glu His Pro Val Thr Glu Cys Ile Thr Gly Leu Asp

355 360 365

Leu Val Gln Glu Met Ile Arg Val Ala Lys Gly Tyr Pro Leu Arg His

370 375 380

Lys Gln Ala Asp Ile Arg Ile Asn Gly Trp Ala Val Glu Cys Arg Val

385 390 395 400

Tyr Ala Glu Asp Pro Tyr Lys Ser Phe Gly Leu Pro Ser Ile Gly Arg

405 410 415

Leu Ser Gln Tyr Gln Glu Pro Leu His Leu Pro Gly Val Arg Val Asp

420 425 430

Ser Gly Ile Gln Pro Gly Ser Asp Ile Ser Ile Tyr Tyr Asp Pro Met

435 440 445

Ile Ser Lys Leu Ile Thr Tyr Gly Ser Asp Arg Thr Glu Ala Leu Lys

450 455 460

Arg Met Ala Asp Ala Leu Asp Asn Tyr Val Ile Arg Gly Val Thr His

465 470 475 480

Asn Ile Ala Leu Leu Arg Glu Val Ile Ile Asn Ser Arg Phe Val Lys

485 490 495

Gly Asp Ile Ser Thr Lys Phe Leu Ser Asp Val Tyr Pro Asp Gly Phe

500 505 510

Lys Gly His Met Leu Thr Lys Ser Glu Lys Asn Gln Leu Leu Ala Ile

515 520 525

Ala Ser Ser Leu Phe Val Ala Phe Gln Leu Arg Ala Gln His Phe Gln

530 535 540

Glu Asn Ser Arg Met Pro Val Ile Lys Pro Asp Ile Ala Asn Trp Glu

545 550 555 560

Leu Ser Val Lys Leu His Asp Lys Val His Thr Val Val Ala Ser Asn

565 570 575

Asn Gly Ser Val Phe Ser Val Glu Val Asp Gly Ser Lys Leu Asn Val

580 585 590

Thr Ser Thr Trp Asn Leu Ala Ser Pro Leu Leu Ser Val Ser Val Asp

595 600 605

Gly Thr Gln Arg Thr Val Gln Cys Leu Ser Arg Glu Ala Gly Gly Asn

610 615 620

Met Ser Ile Gln Phe Leu Gly Thr Val Tyr Lys Val Asn Ile Leu Thr

625 630 635 640

Arg Leu Ala Ala Glu Leu Asn Lys Phe Met Leu Glu Lys Val Thr Glu

645 650 655

Asp Thr Ser Ser Val Leu Arg Ser Pro Met Pro Gly Val Val Val Ala

660 665 670

Val Ser Val Lys Pro Gly Asp Ala Val Ala Glu Gly Gln Glu Ile Cys

675 680 685

Val Ile Glu Ala Met Lys Met Gln Asn Ser Met Thr Ala Gly Lys Thr

690 695 700

Gly Thr Val Lys Ser Val His Cys Gln Ala Gly Asp Thr Val Gly Glu

705 710 715 720

Gly Asp Leu Leu Val Glu Leu Glu

725

<210> 17

<211> 539

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 17

Met Ala Ala Ala Leu Arg Val Ala Ala Val Gly Ala Arg Leu Ser Val

1 5 10 15

Leu Ala Ser Gly Leu Arg Ala Ala Val Arg Ser Leu Cys Ser Gln Ala

20 25 30

Thr Ser Val Asn Glu Arg Ile Glu Asn Lys Arg Arg Thr Ala Leu Leu

35 40 45

Gly Gly Gly Gln Arg Arg Ile Asp Ala Gln His Lys Arg Gly Lys Leu

50 55 60

Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu Asp Pro Gly Ser Phe Val

65 70 75 80

Glu Ser Asp Met Phe Val Glu His Arg Cys Ala Asp Phe Gly Met Ala

85 90 95

Ala Asp Lys Asn Lys Phe Pro Gly Asp Ser Val Val Thr Gly Arg Gly

100 105 110

Arg Ile Asn Gly Arg Leu Val Tyr Val Phe Ser Gln Asp Phe Thr Val

115 120 125

Phe Gly Gly Ser Leu Ser Gly Ala His Ala Gln Lys Ile Cys Lys Ile

130 135 140

Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile Gly Leu Asn Asp

145 150 155 160

Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser Leu Ala Gly Tyr

165 170 175

Ala Asp Ile Phe Leu Arg Asn Val Thr Ala Ser Gly Val Ile Pro Gln

180 185 190

Ile Ser Leu Ile Met Gly Pro Cys Ala Gly Gly Ala Val Tyr Ser Pro

195 200 205

Ala Leu Thr Asp Phe Thr Phe Met Val Lys Asp Thr Ser Tyr Leu Phe

210 215 220

Ile Thr Gly Pro Asp Val Val Lys Ser Val Thr Asn Glu Asp Val Thr

225 230 235 240

Gln Glu Glu Leu Gly Gly Ala Lys Thr His Thr Thr Met Ser Gly Val

245 250 255

Ala His Arg Ala Phe Glu Asn Asp Val Asp Ala Leu Cys Asn Leu Arg

260 265 270

Asp Phe Phe Asn Tyr Leu Pro Leu Ser Ser Gln Asp Pro Ala Pro Val

275 280 285

Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro Glu Leu Asp Thr

290 295 300

Ile Val Pro Leu Glu Ser Thr Lys Ala Tyr Asn Met Val Asp Ile Ile

305 310 315 320

His Ser Val Val Asp Glu Arg Glu Phe Phe Glu Ile Met Pro Asn Tyr

325 330 335

Ala Lys Asn Ile Ile Val Gly Phe Ala Arg Met Asn Gly Arg Thr Val

340 345 350

Gly Ile Val Gly Asn Gln Pro Lys Val Ala Ser Gly Cys Leu Asp Ile

355 360 365

Asn Ser Ser Val Lys Gly Ala Arg Phe Val Arg Phe Cys Asp Ala Phe

370 375 380

Asn Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly Phe Leu Pro Gly

385 390 395 400

Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly Ala Lys Leu Leu

405 410 415

Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Val Thr Val Ile Thr Arg

420 425 430

Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser Ser Lys His Leu Cys

435 440 445

Gly Asp Thr Asn Tyr Ala Trp Pro Thr Ala Glu Ile Ala Val Met Gly

450 455 460

Ala Lys Gly Ala Val Glu Ile Ile Phe Lys Gly His Glu Asn Val Glu

465 470 475 480

Ala Ala Gln Ala Glu Tyr Ile Glu Lys Phe Ala Asn Pro Phe Pro Ala

485 490 495

Ala Val Arg Gly Phe Val Asp Asp Ile Ile Gln Pro Ser Ser Thr Arg

500 505 510

Ala Arg Ile Cys Cys Asp Leu Asp Val Leu Ala Ser Lys Lys Val Gln

515 520 525

Arg Pro Trp Arg Lys His Ala Asn Ile Pro Leu

530 535

<210> 18

<211> 278

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: CBA promoters

<400> 18

tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60

ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120

ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 180

cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240

ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcg 278

<210> 19

<211> 304

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: CMV enhancer

<400> 19

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catg 304

<210> 20

<211> 95

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: SV40 Small T intron

<400> 20

gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg 60

tttctctctt ttagattcca acctttggaa ctgat 95

<210> 21

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: rHBB intron

<400> 21

gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 60

cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc 120

ggggggagcg gctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag 180

ggcggggttc ggcttctggc gtgtgaccgg cggctcaaga gcctctgcta accatgttca 240

tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca 300

ttttggcaaa gaatt 315

<210> 22

<211> 215

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: BGH polyadenylation signal sequence

<400> 22

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 60

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 120

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 180

gaggattggg aagacaatag caggcatgct gggga 215

<210> 23

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: SV40 late polyadenylation signal sequence

<400> 23

gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 60

aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 120

tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag 180

gtgtgggagg ttttttag 198

<210> 24

<400> 24

000

<210> 25

<211> 728

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 25

Met Ala Gly Phe Trp Val Gly Thr Ala Pro Leu Val Ala Ala Gly Arg

1 5 10 15

Arg Gly Arg Trp Pro Pro Gln Gln Leu Met Leu Ser Ala Ala Leu Arg

20 25 30

Thr Leu Lys His Val Leu Tyr Tyr Ser Arg Gln Cys Leu Met Val Ser

35 40 45

Arg Asn Leu Gly Ser Val Gly Tyr Asp Pro Asn Glu Lys Thr Phe Asp

50 55 60

Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Ala Cys Arg Val Ile Arg

65 70 75 80

Thr Cys Lys Lys Met Gly Ile Lys Thr Val Ala Ile His Ser Asp Val

85 90 95

Asp Ala Ser Ser Val His Val Lys Met Ala Asp Glu Ala Val Cys Val

100 105 110

Gly Pro Ala Pro Thr Ser Lys Ser Tyr Leu Asn Met Asp Ala Ile Met

115 120 125

Glu Ala Ile Lys Lys Thr Arg Ala Gln Ala Val His Pro Gly Tyr Gly

130 135 140

Phe Leu Ser Glu Asn Lys Glu Phe Ala Arg Cys Leu Ala Ala Glu Asp

145 150 155 160

Val Val Phe Ile Gly Pro Asp Thr His Ala Ile Gln Ala Met Gly Asp

165 170 175

Lys Ile Glu Ser Lys Leu Leu Ala Lys Lys Ala Glu Val Asn Thr Ile

180 185 190

Pro Gly Phe Asp Gly Val Val Lys Asp Ala Glu Glu Ala Val Arg Ile

195 200 205

Ala Arg Glu Ile Gly Tyr Pro Val Met Ile Lys Ala Ser Ala Gly Gly

210 215 220

Gly Gly Lys Gly Met Arg Ile Ala Trp Asp Asp Glu Glu Thr Arg Asp

225 230 235 240

Gly Phe Arg Leu Ser Ser Gln Glu Ala Ala Ser Ser Phe Gly Asp Asp

245 250 255

Arg Leu Leu Ile Glu Lys Phe Ile Asp Asn Pro Arg His Ile Glu Ile

260 265 270

Gln Val Leu Gly Asp Lys His Gly Asn Ala Leu Trp Leu Asn Glu Arg

275 280 285

Glu Cys Ser Ile Gln Arg Arg Asn Gln Lys Val Val Glu Glu Ala Pro

290 295 300

Ser Ile Phe Leu Asp Ala Glu Thr Arg Arg Ala Met Gly Glu Gln Ala

305 310 315 320

Val Ala Leu Ala Arg Ala Val Lys Tyr Ser Ser Ala Gly Thr Val Glu

325 330 335

Phe Leu Val Asp Ser Lys Lys Asn Phe Tyr Phe Leu Glu Met Asn Thr

340 345 350

Arg Leu Gln Val Glu His Pro Val Thr Glu Cys Ile Thr Gly Leu Asp

355 360 365

Leu Val Gln Glu Met Ile Arg Val Ala Lys Gly Tyr Pro Leu Arg His

370 375 380

Lys Gln Ala Asp Ile Arg Ile Asn Gly Trp Ala Val Glu Cys Arg Val

385 390 395 400

Tyr Ala Glu Asp Pro Tyr Lys Ser Phe Gly Leu Pro Ser Ile Gly Arg

405 410 415

Leu Ser Gln Tyr Gln Glu Pro Leu His Leu Pro Gly Val Arg Val Asp

420 425 430

Ser Gly Ile Gln Pro Gly Ser Asp Ile Ser Ile Tyr Tyr Asp Pro Met

435 440 445

Ile Ser Lys Leu Ile Thr Tyr Gly Ser Asp Arg Thr Glu Ala Leu Lys

450 455 460

Arg Met Ala Asp Ala Leu Asp Asn Tyr Val Ile Arg Gly Val Thr His

465 470 475 480

Asn Ile Ala Leu Leu Arg Glu Val Ile Ile Asn Ser Arg Phe Val Lys

485 490 495

Gly Asp Ile Ser Thr Lys Phe Leu Ser Asp Val Tyr Pro Asp Gly Phe

500 505 510

Lys Gly His Met Leu Thr Lys Ser Glu Lys Asn Gln Leu Leu Ala Ile

515 520 525

Ala Ser Ser Leu Phe Val Ala Phe Gln Leu Arg Ala Gln His Phe Gln

530 535 540

Glu Asn Ser Arg Met Pro Val Ile Lys Pro Asp Ile Ala Asn Trp Glu

545 550 555 560

Leu Ser Val Lys Leu His Asp Lys Val His Thr Val Val Ala Ser Asn

565 570 575

Asn Gly Ser Val Phe Ser Val Glu Val Asp Gly Ser Lys Leu Asn Val

580 585 590

Thr Ser Thr Trp Asn Leu Ala Ser Pro Leu Leu Ser Val Ser Val Asp

595 600 605

Gly Thr Gln Arg Thr Val Gln Cys Leu Ser Arg Glu Ala Gly Gly Asn

610 615 620

Met Ser Ile Gln Phe Leu Gly Thr Val Tyr Lys Val Asn Ile Leu Thr

625 630 635 640

Arg Leu Ala Ala Glu Leu Asn Lys Phe Met Leu Glu Lys Val Thr Glu

645 650 655

Asp Thr Ser Ser Val Leu Arg Ser Pro Met Pro Gly Val Val Val Ala

660 665 670

Val Ser Val Lys Pro Gly Asp Ala Val Ala Glu Gly Gln Glu Ile Cys

675 680 685

Val Ile Glu Ala Met Lys Met Gln Asn Ser Met Thr Ala Gly Lys Thr

690 695 700

Gly Thr Val Lys Ser Val His Cys Gln Ala Gly Asp Thr Val Gly Glu

705 710 715 720

Gly Asp Leu Leu Val Glu Leu Glu

725

<210> 26

<211> 702

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 26

Met Ala Gly Phe Trp Val Gly Thr Ala Pro Leu Val Ala Ala Gly Arg

1 5 10 15

Arg Gly Arg Trp Pro Pro Gln Gln Leu Met Leu Ser Ala Ala Leu Arg

20 25 30

Thr Leu Lys Thr Phe Asp Lys Ile Leu Val Ala Asn Arg Gly Glu Ile

35 40 45

Ala Cys Arg Val Ile Arg Thr Cys Lys Lys Met Gly Ile Lys Thr Val

50 55 60

Ala Ile His Ser Asp Val Asp Ala Ser Ser Val His Val Lys Met Ala

65 70 75 80

Asp Glu Ala Val Cys Val Gly Pro Ala Pro Thr Ser Lys Ser Tyr Leu

85 90 95

Asn Met Asp Ala Ile Met Glu Ala Ile Lys Lys Thr Arg Ala Gln Ala

100 105 110

Val His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Lys Glu Phe Ala Arg

115 120 125

Cys Leu Ala Ala Glu Asp Val Val Phe Ile Gly Pro Asp Thr His Ala

130 135 140

Ile Gln Ala Met Gly Asp Lys Ile Glu Ser Lys Leu Leu Ala Lys Lys

145 150 155 160

Ala Glu Val Asn Thr Ile Pro Gly Phe Asp Gly Val Val Lys Asp Ala

165 170 175

Glu Glu Ala Val Arg Ile Ala Arg Glu Ile Gly Tyr Pro Val Met Ile

180 185 190

Lys Ala Ser Ala Gly Gly Gly Gly Lys Gly Met Arg Ile Ala Trp Asp

195 200 205

Asp Glu Glu Thr Arg Asp Gly Phe Arg Leu Ser Ser Gln Glu Ala Ala

210 215 220

Ser Ser Phe Gly Asp Asp Arg Leu Leu Ile Glu Lys Phe Ile Asp Asn

225 230 235 240

Pro Arg His Ile Glu Ile Gln Val Leu Gly Asp Lys His Gly Asn Ala

245 250 255

Leu Trp Leu Asn Glu Arg Glu Cys Ser Ile Gln Arg Arg Asn Gln Lys

260 265 270

Val Val Glu Glu Ala Pro Ser Ile Phe Leu Asp Ala Glu Thr Arg Arg

275 280 285

Ala Met Gly Glu Gln Ala Val Ala Leu Ala Arg Ala Val Lys Tyr Ser

290 295 300

Ser Ala Gly Thr Val Glu Phe Leu Val Asp Ser Lys Lys Asn Phe Tyr

305 310 315 320

Phe Leu Glu Met Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu

325 330 335

Cys Ile Thr Gly Leu Asp Leu Val Gln Glu Met Ile Arg Val Ala Lys

340 345 350

Gly Tyr Pro Leu Arg His Lys Gln Ala Asp Ile Arg Ile Asn Gly Trp

355 360 365

Ala Val Glu Cys Arg Val Tyr Ala Glu Asp Pro Tyr Lys Ser Phe Gly

370 375 380

Leu Pro Ser Ile Gly Arg Leu Ser Gln Tyr Gln Glu Pro Leu His Leu

385 390 395 400

Pro Gly Val Arg Val Asp Ser Gly Ile Gln Pro Gly Ser Asp Ile Ser

405 410 415

Ile Tyr Tyr Asp Pro Met Ile Ser Lys Leu Ile Thr Tyr Gly Ser Asp

420 425 430

Arg Thr Glu Ala Leu Lys Arg Met Ala Asp Ala Leu Asp Asn Tyr Val

435 440 445

Ile Arg Gly Val Thr His Asn Ile Ala Leu Leu Arg Glu Val Ile Ile

450 455 460

Asn Ser Arg Phe Val Lys Gly Asp Ile Ser Thr Lys Phe Leu Ser Asp

465 470 475 480

Val Tyr Pro Asp Gly Phe Lys Gly His Met Leu Thr Lys Ser Glu Lys

485 490 495

Asn Gln Leu Leu Ala Ile Ala Ser Ser Leu Phe Val Ala Phe Gln Leu

500 505 510

Arg Ala Gln His Phe Gln Glu Asn Ser Arg Met Pro Val Ile Lys Pro

515 520 525

Asp Ile Ala Asn Trp Glu Leu Ser Val Lys Leu His Asp Lys Val His

530 535 540

Thr Val Val Ala Ser Asn Asn Gly Ser Val Phe Ser Val Glu Val Asp

545 550 555 560

Gly Ser Lys Leu Asn Val Thr Ser Thr Trp Asn Leu Ala Ser Pro Leu

565 570 575

Leu Ser Val Ser Val Asp Gly Thr Gln Arg Thr Val Gln Cys Leu Ser

580 585 590

Arg Glu Ala Gly Gly Asn Met Ser Ile Gln Phe Leu Gly Thr Val Tyr

595 600 605

Lys Val Asn Ile Leu Thr Arg Leu Ala Ala Glu Leu Asn Lys Phe Met

610 615 620

Leu Glu Lys Val Thr Glu Asp Thr Ser Ser Val Leu Arg Ser Pro Met

625 630 635 640

Pro Gly Val Val Val Ala Val Ser Val Lys Pro Gly Asp Ala Val Ala

645 650 655

Glu Gly Gln Glu Ile Cys Val Ile Glu Ala Met Lys Met Gln Asn Ser

660 665 670

Met Thr Ala Gly Lys Thr Gly Thr Val Lys Ser Val His Cys Gln Ala

675 680 685

Gly Asp Thr Val Gly Glu Gly Asp Leu Leu Val Glu Leu Glu

690 695 700

<210> 27

<211> 681

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 27

Met Ala Gly Phe Trp Val Gly Thr Ala Pro Leu Val Ala Ala Gly Arg

1 5 10 15

Arg Gly Arg Trp Pro Pro Gln Gln Leu Met Leu Ser Ala Ala Leu Arg

20 25 30

Thr Leu Lys His Val Leu Tyr Tyr Ser Arg Gln Cys Leu Met Val Ser

35 40 45

Arg Asn Leu Gly Ser Val Gly Tyr Asp Pro Asn Glu Lys Thr Phe Asp

50 55 60

Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Ala Cys Arg Val Ile Arg

65 70 75 80

Thr Cys Lys Lys Met Gly Ile Lys Thr Val Ala Ile His Ser Asp Val

85 90 95

Asp Ala Ser Ser Val His Val Lys Met Ala Asp Glu Ala Val Cys Val

100 105 110

Gly Pro Ala Pro Thr Ser Lys Ser Tyr Leu Asn Met Asp Ala Ile Met

115 120 125

Glu Ala Ile Lys Lys Thr Arg Ala Gln Ala Val His Pro Gly Tyr Gly

130 135 140

Phe Leu Ser Glu Asn Lys Glu Phe Ala Arg Cys Leu Ala Ala Glu Asp

145 150 155 160

Val Val Phe Ile Gly Pro Asp Thr His Ala Ile Gln Ala Met Gly Asp

165 170 175

Lys Ile Glu Ser Lys Leu Leu Ala Lys Lys Ala Glu Val Asn Thr Ile

180 185 190

Pro Gly Phe Asp Gly Val Val Lys Asp Ala Glu Glu Ala Val Arg Ile

195 200 205

Ala Arg Glu Ile Gly Tyr Pro Val Met Ile Lys Ala Ser Ala Gly Gly

210 215 220

Gly Gly Lys Gly Met Arg Ile Ala Trp Asp Asp Glu Glu Thr Arg Asp

225 230 235 240

Gly Phe Arg Leu Ser Ser Gln Glu Ala Ala Ser Ser Phe Gly Asp Asp

245 250 255

Arg Leu Leu Ile Glu Lys Phe Ile Asp Asn Pro Arg His Ile Glu Ile

260 265 270

Gln Val Leu Gly Asp Lys His Gly Asn Ala Leu Trp Leu Asn Glu Arg

275 280 285

Glu Cys Ser Ile Gln Arg Arg Asn Gln Lys Val Val Glu Glu Ala Pro

290 295 300

Ser Ile Phe Leu Asp Ala Glu Thr Arg Arg Ala Met Gly Glu Gln Ala

305 310 315 320

Val Ala Leu Ala Arg Ala Val Lys Tyr Ser Ser Ala Gly Thr Val Glu

325 330 335

Phe Leu Val Asp Ser Lys Lys Asn Phe Tyr Phe Leu Glu Met Asn Thr

340 345 350

Arg Leu Gln Val Glu His Pro Val Thr Glu Cys Ile Thr Gly Leu Asp

355 360 365

Leu Val Gln Glu Met Ile Arg Val Ala Lys Gly Tyr Pro Leu Arg His

370 375 380

Lys Gln Ala Asp Ile Arg Ile Asn Gly Trp Ala Val Glu Cys Arg Val

385 390 395 400

Tyr Ala Glu Asp Pro Tyr Lys Ser Phe Gly Leu Pro Ser Ile Gly Arg

405 410 415

Leu Ser Gln Tyr Gln Glu Pro Leu His Leu Pro Gly Val Arg Val Asp

420 425 430

Ser Gly Ile Gln Pro Gly Ser Asp Ile Ser Ile Tyr Tyr Asp Pro Met

435 440 445

Ile Ser Lys Leu Ile Thr Tyr Gly Ser Asp Arg Thr Glu Ala Leu Lys

450 455 460

Arg Met Ala Asp Ala Leu Asp Asn Tyr Val Ile Arg Gly Val Thr His

465 470 475 480

Asn Ile Ala Leu Leu Arg Glu Val Ile Ile Asn Ser Arg Phe Val Lys

485 490 495

Gly Asp Ile Ser Thr Lys Phe Leu Ser Asp Val Tyr Pro Asp Gly Phe

500 505 510

Lys Gly His Met Leu Thr Lys Ser Glu Lys Asn Gln Leu Leu Ala Ile

515 520 525

Ala Ser Ser Leu Phe Val Ala Phe Gln Leu Arg Ala Gln His Phe Gln

530 535 540

Glu Asn Ser Arg Met Pro Val Ile Lys Pro Asp Ile Ala Asn Trp Glu

545 550 555 560

Leu Ser Val Lys Leu His Asp Lys Val His Thr Val Val Ala Ser Asn

565 570 575

Asn Gly Ser Val Phe Ser Val Glu Val Asp Gly Ser Lys Leu Asn Val

580 585 590

Thr Ser Thr Trp Asn Leu Ala Ser Pro Leu Leu Ser Val Ser Val Asp

595 600 605

Gly Thr Gln Arg Thr Val Gln Cys Leu Ser Arg Glu Ala Gly Gly Asn

610 615 620

Met Ser Ile Gln Phe Leu Gly Thr Val Val Ala Glu Gly Gln Glu Ile

625 630 635 640

Cys Val Ile Glu Ala Met Lys Met Gln Asn Ser Met Thr Ala Gly Lys

645 650 655

Thr Gly Thr Val Lys Ser Val His Cys Gln Ala Gly Asp Thr Val Gly

660 665 670

Glu Gly Asp Leu Leu Val Glu Leu Glu

675 680

<210> 28

<211> 539

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 28

Met Ala Ala Ala Leu Arg Val Ala Ala Val Gly Ala Arg Leu Ser Val

1 5 10 15

Leu Ala Ser Gly Leu Arg Ala Ala Val Arg Ser Leu Cys Ser Gln Ala

20 25 30

Thr Ser Val Asn Glu Arg Ile Glu Asn Lys Arg Arg Thr Ala Leu Leu

35 40 45

Gly Gly Gly Gln Arg Arg Ile Asp Ala Gln His Lys Arg Gly Lys Leu

50 55 60

Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu Asp Pro Gly Ser Phe Val

65 70 75 80

Glu Ser Asp Met Phe Val Glu His Arg Cys Ala Asp Phe Gly Met Ala

85 90 95

Ala Asp Lys Asn Lys Phe Pro Gly Asp Ser Val Val Thr Gly Arg Gly

100 105 110

Arg Ile Asn Gly Arg Leu Val Tyr Val Phe Ser Gln Asp Phe Thr Val

115 120 125

Phe Gly Gly Ser Leu Ser Gly Ala His Ala Gln Lys Ile Cys Lys Ile

130 135 140

Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile Gly Leu Asn Asp

145 150 155 160

Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser Leu Ala Gly Tyr

165 170 175

Ala Asp Ile Phe Leu Arg Asn Val Thr Ala Ser Gly Val Ile Pro Gln

180 185 190

Ile Ser Leu Ile Met Gly Pro Cys Ala Gly Gly Ala Val Tyr Ser Pro

195 200 205

Ala Leu Thr Asp Phe Thr Phe Met Val Lys Asp Thr Ser Tyr Leu Phe

210 215 220

Ile Thr Gly Pro Asp Val Val Lys Ser Val Thr Asn Glu Asp Val Thr

225 230 235 240

Gln Glu Glu Leu Gly Gly Ala Lys Thr His Thr Thr Met Ser Gly Val

245 250 255

Ala His Arg Ala Phe Glu Asn Asp Val Asp Ala Leu Cys Asn Leu Arg

260 265 270

Asp Phe Phe Asn Tyr Leu Pro Leu Ser Ser Gln Asp Pro Ala Pro Val

275 280 285

Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro Glu Leu Asp Thr

290 295 300

Ile Val Pro Leu Glu Ser Thr Lys Ala Tyr Asn Met Val Asp Ile Ile

305 310 315 320

His Ser Val Val Asp Glu Arg Glu Phe Phe Glu Ile Met Pro Asn Tyr

325 330 335

Ala Lys Asn Ile Ile Val Gly Phe Ala Arg Met Asn Gly Arg Thr Val

340 345 350

Gly Ile Val Gly Asn Gln Pro Lys Val Ala Ser Gly Cys Leu Asp Ile

355 360 365

Asn Ser Ser Val Lys Gly Ala Arg Phe Val Arg Phe Cys Asp Ala Phe

370 375 380

Asn Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly Phe Leu Pro Gly

385 390 395 400

Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly Ala Lys Leu Leu

405 410 415

Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Val Thr Val Ile Thr Arg

420 425 430

Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser Ser Lys His Leu Cys

435 440 445

Gly Asp Thr Asn Tyr Ala Trp Pro Thr Ala Glu Ile Ala Val Met Gly

450 455 460

Ala Lys Gly Ala Val Glu Ile Ile Phe Lys Gly His Glu Asn Val Glu

465 470 475 480

Ala Ala Gln Ala Glu Tyr Ile Glu Lys Phe Ala Asn Pro Phe Pro Ala

485 490 495

Ala Val Arg Gly Phe Val Asp Asp Ile Ile Gln Pro Ser Ser Thr Arg

500 505 510

Ala Arg Ile Cys Cys Asp Leu Asp Val Leu Ala Ser Lys Lys Val Gln

515 520 525

Arg Pro Trp Arg Lys His Ala Asn Ile Pro Leu

530 535

<210> 29

<211> 559

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 29

Met Ala Ala Ala Leu Arg Val Ala Ala Val Gly Ala Arg Leu Ser Val

1 5 10 15

Leu Ala Ser Gly Leu Arg Ala Ala Val Arg Ser Leu Cys Ser Gln Ala

20 25 30

Thr Ser Val Asn Glu Arg Ile Glu Asn Lys Arg Arg Thr Ala Leu Leu

35 40 45

Gly Gly Gly Gln Arg Arg Ile Asp Ala Gln His Lys Arg Gly Lys Leu

50 55 60

Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu Asp Pro Gly Ser Phe Val

65 70 75 80

Glu Ser Asp Met Phe Val Glu His Arg Cys Ala Asp Phe Gly Met Ala

85 90 95

Ala Asp Lys Asn Lys Phe Pro Gly Asp Ser Val Val Thr Gly Arg Gly

100 105 110

Arg Ile Asn Gly Arg Leu Val Tyr Val Phe Ser Gln Gln Ile Ile Gly

115 120 125

Trp Ala Gln Trp Leu Pro Leu Val Ile Ser Ala Leu Trp Glu Ala Glu

130 135 140

Asp Phe Thr Val Phe Gly Gly Ser Leu Ser Gly Ala His Ala Gln Lys

145 150 155 160

Ile Cys Lys Ile Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile

165 170 175

Gly Leu Asn Asp Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser

180 185 190

Leu Ala Gly Tyr Ala Asp Ile Phe Leu Arg Asn Val Thr Ala Ser Gly

195 200 205

Val Ile Pro Gln Ile Ser Leu Ile Met Gly Pro Cys Ala Gly Gly Ala

210 215 220

Val Tyr Ser Pro Ala Leu Thr Asp Phe Thr Phe Met Val Lys Asp Thr

225 230 235 240

Ser Tyr Leu Phe Ile Thr Gly Pro Asp Val Val Lys Ser Val Thr Asn

245 250 255

Glu Asp Val Thr Gln Glu Glu Leu Gly Gly Ala Lys Thr His Thr Thr

260 265 270

Met Ser Gly Val Ala His Arg Ala Phe Glu Asn Asp Val Asp Ala Leu

275 280 285

Cys Asn Leu Arg Asp Phe Phe Asn Tyr Leu Pro Leu Ser Ser Gln Asp

290 295 300

Pro Ala Pro Val Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro

305 310 315 320

Glu Leu Asp Thr Ile Val Pro Leu Glu Ser Thr Lys Ala Tyr Asn Met

325 330 335

Val Asp Ile Ile His Ser Val Val Asp Glu Arg Glu Phe Phe Glu Ile

340 345 350

Met Pro Asn Tyr Ala Lys Asn Ile Ile Val Gly Phe Ala Arg Met Asn

355 360 365

Gly Arg Thr Val Gly Ile Val Gly Asn Gln Pro Lys Val Ala Ser Gly

370 375 380

Cys Leu Asp Ile Asn Ser Ser Val Lys Gly Ala Arg Phe Val Arg Phe

385 390 395 400

Cys Asp Ala Phe Asn Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly

405 410 415

Phe Leu Pro Gly Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly

420 425 430

Ala Lys Leu Leu Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Val Thr

435 440 445

Val Ile Thr Arg Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser Ser

450 455 460

Lys His Leu Cys Gly Asp Thr Asn Tyr Ala Trp Pro Thr Ala Glu Ile

465 470 475 480

Ala Val Met Gly Ala Lys Gly Ala Val Glu Ile Ile Phe Lys Gly His

485 490 495

Glu Asn Val Glu Ala Ala Gln Ala Glu Tyr Ile Glu Lys Phe Ala Asn

500 505 510

Pro Phe Pro Ala Ala Val Arg Gly Phe Val Asp Asp Ile Ile Gln Pro

515 520 525

Ser Ser Thr Arg Ala Arg Ile Cys Cys Asp Leu Asp Val Leu Ala Ser

530 535 540

Lys Lys Val Gln Arg Pro Trp Arg Lys His Ala Asn Ile Pro Leu

545 550 555

<210> 30

<211> 1412

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 30

tctcaaggtg actctgcagg agatgcaaag tagttaagag gagaaccccc aatactgatg 60

tgtttatagg tgaggtggtc tgatgtctta gatttgcttt aaaatattcc tgcacaaaat 120

aaaaagggga atagatgaaa caagattgac aaaatgttga ttcattgacg cggtgtaatg 180

ggtatctggg tgatcatagt acaattttat ctcttttgta tatattttaa aatttccata 240

atatagcaaa acatgagccc agtccctcaa ggcagccaga tttgggttca aaccctagtt 300

tgacctttta ctcgctgagt gactttaccc tgctacagct tcagtttctc caaatggaaa 360

taatatatac tccattaagg ttcttgtaaa ctctcaagta agagggaatc tgaggctcta 420

gcacacggcc cggcccttaa tagttgtact aatttttatt ttatttttcg aaacggagtt 480

tgctctgtca cccaggctgg agtgtagtgg catgatccgg ctcactgcaa cctccacctc 540

cagggttcaa gcaattctcc tgcctcagcc tcctcccgag taactgggat tacaggagtg 600

cgccaccacg cccagctaat ttttgtattt tttagtagag acggggtttc ggcatgttgt 660

ccaggctggt ctccaactcc tgacctcagg ttatcctccc gcctcggcct cccagagtgc 720

tgggattaca ggcgtgagcc actgcgcctg gccagtagtt gtactaacaa gggtttctgc 780

ttgcgagcaa caaagactga ctctagttgt ggctaatttc tttcacacac acatattaag 840

tgccagaggt aggacttgag ctcggatctc tgatttccag acctagaact ttttatgtgt 900

tattacagtc gttaaaaacc ttacctggat ttgtgctttg caatttacgc tttcgaacat 960

gcaggaatgt agtttgggca tttgtctgga acttcggatt ctcgggactc cagggtggcc 1020

aaggcgcgga ggtgactcaa tgacattgac cggcctggat gcacgtgggt tgctgagccg 1080

atgctgggaa ggtacggggt ttgcccggtg caggccgccg gaactcacgc acgcccctgt 1140

ctctctcact cctggggtcg gctctggccg gtggcctcca gctcagctag cggccggacg 1200

cgcagcccgc cgactagccc tccaggtcct agcgcgactc ccggatcccg gatccggcgg 1260

atctggccga ccactcaccc tcccctttcc gcaagttagg gggcggggca tcgggtttct 1320

ggctcgtgat ttgccggagc tcctgcgctc cccttctcca ccccctccgg ctgtgtgaga 1380

ggtcagcaga ggggcggtct gcggggacaa ca 1412

<210> 31

<211> 275

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 31

aggatttata acctttcagt catcacccaa tttaattagc catttgcatg atgctttcac 60

acacaattga ttcaagcatt atacaggaac acccctgtgc agctacgttt acgtcgtcat 120

ttattccaca gagtcaagac caatattctg ccaaaaaatc accaatggaa attttcattg 180

atataaatac ttgtacatat gatttgtact tctgctgtga gattccctag tgtcaaaatt 240

aaatcaataa aactgagcat ttgtctaaat attag 275

<210> 32

<211> 1378

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 32

gctggcccca gccccagagt ttctgattta aaggtctgga gtaagttctg agaattcgag 60

cttgcatttc caactcactc ccaggtgata ctgaagttgc tggattgaca agattccctc 120

cttgatcaaa cattggtcag gctcttctga gctctctctt tgactaggcc tgagtgtggg 180

ctcccatgtc attccttgtg gaattccatt ttagcaagaa acttgccaag tcagtttagc 240

cagaatcctc cattcttgat atctgattac tctccatatc tggttttgac ccataccgcc 300

accatccccc aggtgatgtt cagttaccct gacctgcctt gggcaggaat cctgttaggt 360

caatttagcc agattccccg ccaatgtttc ctcttagtaa ttttccatcc actgacttcc 420

acacactgcc ccttagctat aaattaccac tttgctatat tcagcccaat cgctctgact 480

tatggcaaaa ctctatccct acctctgtca gattgggcct gaataaagtt tgccttacca 540

ttcttaaaca tgaataatta agtgtcatga atagtttttt ctttaatggg atccatactt 600

tgagaactac tgattagagg ttaagggtcc tgtgctcatt atctggcaca tattaatgat 660

aggtctctga gtttgaatcc tggccaacca cattgtcagt cttggctttt tttttttttt 720

ttttttgaga cagggttttg cccaggctgg agtgcagtga tgcaatcacg gctcactgca 780

gcctcgactt cctgggccaa gccatcctgc cgccttagcc tcccgagtag ctggggtcac 840

aggtgcaagc cgccatgccc agctaatttt taattttttt gtagaggtgg ggtcttacta 900

tattgctcag gctggtcttg ggctcctggg ctcacctacc tcggcctcag tcttgcctct 960

tgatcaaatc actcatctct ctgtgcttga tttacctcat ctgtgaaatc cagatagtaa 1020

gctagtgcat ggtaagtgca aaaagcaaca cctggcacaa tgcttaataa atgttggtta 1080

aatcttataa tcattttctg taggaagtgg gacgacaaag ttaagggtct aggtggtcag 1140

agaagagcaa ggactatgtc ccttttttgt gcagccaggc aggccctgta ctttttctct 1200

gcgcgaccct cctcgggcag caccgggtct cgaactctca cgccttactc ccaagcaggt 1260

tctgtagtcc agcccccgcg gctccctaca gccagccgat gctgcgcaag cgccgtaccc 1320

acgctttagc acatgcgtac tcaggtgcgc cggtagggga cgcgccggca cagcaaaa 1378

<210> 33

<211> 159

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 33

acaaatcaaa ggaaaagaaa ccaagaactg aattactgtc tgcccattca catcccattc 60

ctgccttttg caatcatgaa acctgggaat ccaaatagtt ggataactta gaataactaa 120

gtttattaaa ttctagaaag atctcttttg tgccttact 159

<210> 34

<211> 793

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 34

tttttgtatt ttttagtaga gacggggttt cggcatgttg tccaggctgg tctccaactc 60

ctgacctcag gttatcctcc cgcctcggcc tcccagagtg ctgggattac aggcgtgagc 120

cactgcgcct ggccagtagt tgtactaaca agggtttctg cttgcgagca acaaagactg 180

actctagttg tggctaattt ctttcacaca cacatattaa gtgccagagg taggacttga 240

gctcggatct ctgatttcca gacctagaac tttttatgtg ttattacagt cgttaaaaac 300

cttacctgga tttgtgcttt gcaatttacg ctttcgaaca tgcaggaatg tagtttgggc 360

atttgtctgg aacttcggat tctcgggact ccagggtggc caaggcgcgg aggtgactca 420

atgacattga ccggcctgga tgcacgtggg ttgctgagcc gatgctggga aggtacgggg 480

tttgcccggt gcaggccgcc ggaactcacg cacgcccctg tctctctcac tcctggggtc 540

ggctctggcc ggtggcctcc agctcagcta gcggccggac gcgcagcccg ccgactagcc 600

ctccaggtcc tagcgcgact cccggatccc ggatccggcg gatctggccg accactcacc 660

ctcccctttc cgcaagttag ggggcggggc atcgggtttc tggctcgtga tttgccggag 720

ctcctgcgct ccccttctcc accccctccg gctgtgtgag aggtcagcag aggggcggtc 780

tgcggggaca aca 793

<210> 35

<211> 168

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 35

aggatttata acctttcagt catcacccaa tttaattagc catttgcatg atgctttcac 60

acacaattga ttcaagcatt atacaggaac acccctgtgc agctacgttt acgtcgtcat 120

ttattccaca gagtcaagac caatattctg ccaaaaaatc accaatgg 168

<210> 36

<211> 468

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 36

gctggtcttg ggctcctggg ctcacctacc tcggcctcag tcttgcctct tgatcaaatc 60

actcatctct ctgtgcttga tttacctcat ctgtgaaatc cagatagtaa gctagtgcat 120

ggtaagtgca aaaagcaaca cctggcacaa tgcttaataa atgttggtta aatcttataa 180

tcattttctg taggaagtgg gacgacaaag ttaagggtct aggtggtcag agaagagcaa 240

ggactatgtc ccttttttgt gcagccaggc aggccctgta ctttttctct gcgcgaccct 300

cctcgggcag caccgggtct cgaactctca cgccttactc ccaagcaggt tctgtagtcc 360

agcccccgcg gctccctaca gccagccgat gctgcgcaag cgccgtaccc acgctttagc 420

acatgcgtac tcaggtgcgc cggtagggga cgcgccggca cagcaaaa 468

<210> 37

<211> 102

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 37

acaaatcaaa ggaaaagaaa ccaagaactg aattactgtc tgcccattca catcccattc 60

ctgccttttg caatcatgaa acctgggaat ccaaatagtt gg 102

<210> 38

<211> 3360

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: rAAV with native PCCA cDNA sequence (DTC346)

<400> 38

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctcgtta cataacttac ggtaaatggc ccgcctggct 180

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 240

caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 300

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 360

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 420

tctacgtatt agtcatcgct attaccatgc gtcgaggtga gccccacgtt ctgcttcact 480

ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt 540

tatgcagcga tgggggcggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga 600

ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg 660

aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg 720

gcgggcggct ctaaggtaaa tataaaattt ttaagtgtat aatgtgttaa actactgatt 780

ctaattgttt ctctctttta gattccaacc tttggaactg atgccgccac catggcgggg 840

ttctgggtcg ggacagcacc gctggtcgct gccggacggc gtgggcggtg gccgccgcag 900

cagctgatgc tgagcgcggc gctgcggacc ctgaagcatg ttctgtacta ttcaagacag 960

tgcttaatgg tgtcccgtaa tcttggttca gtgggatatg atcctaatga aaaaactttt 1020

gataaaattc ttgttgctaa tagaggagaa attgcatgtc gggttattag aacttgcaag 1080

aagatgggca ttaagacagt tgccatccac agtgatgttg atgctagttc tgttcatgtg 1140

aaaatggcgg atgaggctgt ctgtgttggc ccagctccca ccagtaaaag ctacctcaac 1200

atggatgcca tcatggaagc cattaagaaa accagggccc aagctgtaca tccaggttat 1260

ggattccttt cagaaaacaa agaatttgcc agatgtttgg cagcagaaga tgtcgttttc 1320

attggacctg acacacatgc tattcaagcc atgggcgaca agattgaaag caaattatta 1380

gctaagaaag cagaggttaa tacaatccct ggctttgatg gagtagtcaa ggatgcagaa 1440

gaagctgtca gaattgcaag ggaaattggc taccctgtca tgatcaaggc ctcagcaggt 1500

ggtggtggga aaggcatgcg cattgcttgg gatgatgaag agaccaggga tggttttaga 1560

ttgtcatctc aagaagctgc ttctagtttt ggcgatgata gactactaat agaaaaattt 1620

attgataatc ctcgtcatat agaaatccag gttctaggtg ataaacatgg gaatgcttta 1680

tggcttaatg aaagagagtg ctcaattcag agaagaaatc agaaggtggt ggaggaagca 1740

ccaagcattt ttttggatgc ggagactcga agagcgatgg gagaacaagc tgtagctctt 1800

gccagagcag taaaatattc ctctgctggg accgtggagt tccttgtgga ctctaagaag 1860

aatttttatt tcttggaaat gaatacaaga ctccaggttg agcatcctgt cacagaatgc 1920

attactggcc tggacctagt ccaggaaatg atccgtgttg ctaagggcta ccctctcagg 1980

cacaaacaag ctgatattcg catcaacggc tgggcagttg aatgtcgggt ttatgctgag 2040

gacccctaca agtcttttgg tttaccatct attgggagat tgtctcagta ccaagaaccg 2100

ttacatctac ctggtgtccg agtggacagt ggcatccaac caggaagtga tattagcatt 2160

tattatgatc ctatgatttc aaaactaatc acatatggct ctgatagaac tgaggcactg 2220

aagagaatgg cagatgcact ggataactat gttattcgag gtgttacaca taatattgca 2280

ttacttcgag aggtgataat caactcacgc tttgtaaaag gagacatcag cactaaattt 2340

ctctccgatg tgtatcctga tggcttcaaa ggacacatgc taaccaagag tgagaagaac 2400

cagttattgg caatagcatc atcattgttt gtggcattcc agttaagagc acaacatttt 2460

caagaaaatt caagaatgcc tgttattaaa ccagacatag ccaactggga gctctcagta 2520

aaattgcatg ataaagttca taccgtagta gcatcaaaca atgggtcagt gttctcggtg 2580

gaagttgatg ggtcgaaact aaatgtgacc agcacgtgga acctggcttc gcccttattg 2640

tctgtcagcg ttgatggcac tcagaggact gtccagtgtc tttctcgaga agcaggtgga 2700

aacatgagca ttcagtttct tggtacagtg tacaaggtga atatcttaac cagacttgcc 2760

gcagaattga acaaatttat gctggaaaaa gtgactgagg acacaagcag tgttctgcgt 2820

tccccgatgc ccggagtggt ggtggccgtc tctgtcaagc ctggagacgc ggtagcagaa 2880

ggtcaagaaa tttgtgtgat tgaagccatg aaaatgcaga atagtatgac agctgggaaa 2940

actggcacgg tgaaatctgt gcactgtcaa gctggagaca cagttggaga aggggatctg 3000

ctcgtggagc tggaatgaat ccagacatga taagatacat tgatgagttt ggacaaacca 3060

caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 3120

ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 3180

ttcaggttca gggggaggtg tgggaggttt tttagaggaa cccctagtga tggagttggc 3240

cactccctct ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg 3300

ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa 3360

<210> 39

<211> 2806

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: rAAV with native PCCB cDNA sequence

<400> 39

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctcgtta cataacttac ggtaaatggc ccgcctggct 180

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 240

caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 300

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 360

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 420

tctacgtatt agtcatcgct attaccatgt cgaggtgagc cccacgttct gcttcactct 480

ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 540

tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga 600

ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg 660

aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg 720

gcgggcggct ctaaggtaaa tataaaattt ttaagtgtat aatgtgttaa actactgatt 780

ctaattgttt ctctctttta gattccaacc tttggaactg atctcgaggc cgccaccatg 840

gcggcggcat tacgggtggc ggcggtcggg gcaaggctca gcgttctggc gagcggtctc 900

cgcgccgcgg tccgcagcct ttgcagccag gccacctctg ttaacgaacg catcgaaaac 960

aagcgccgga ccgcgctgct gggagggggc caacgccgta ttgacgcgca gcacaagcga 1020

ggaaagctaa cagccaggga gaggatcagt ctcttgctgg accctggcag ctttgttgag 1080

agcgacatgt ttgtggaaca cagatgtgca gattttggaa tggctgctga taagaataag 1140

tttcctggag acagcgtggt cactggacga ggccgaatca atggaagatt ggtttatgtc 1200

ttcagtcagg attttacagt ttttggaggc agtctgtcag gagcacatgc ccaaaagatc 1260

tgcaaaatca tggaccaggc cataacggtg ggggctccag tgattgggct gaatgactct 1320

gggggagcac ggatccaaga aggagtggag tctttggctg gctatgcaga catctttctg 1380

aggaatgtta cggcatccgg agtcatccct cagatttctc tgatcatggg cccatgtgct 1440

ggtggggccg tctactcccc agccctaaca gacttcacgt tcatggtaaa ggacacctcc 1500

tacctgttca tcactggccc tgatgttgtg aagtctgtca ccaatgagga tgttacccag 1560

gaggagctcg gtggtgccaa gacccacacc accatgtcag gtgtggccca cagagctttt 1620

gaaaatgatg ttgatgcctt gtgtaatctc cgggatttct tcaactacct gcccctgagc 1680

agtcaggacc cggctcccgt ccgtgagtgc cacgatccca gtgaccgtct ggttcctgag 1740

cttgacacaa ttgtcccttt ggaatcaacc aaagcctaca acatggtgga catcatacac 1800

tctgttgttg atgagcgtga attttttgag atcatgccca attatgccaa gaacatcatt 1860

gttggttttg caagaatgaa tgggaggact gttggaattg ttggcaacca acctaaggtg 1920

gcctcaggat gcttggatat taattcatct gtgaaagggg ctcgttttgt cagattctgt 1980

gatgcattca atattccact catcactttt gttgatgtcc ctggctttct acctggcaca 2040

gcacaggaat acgggggcat catccggcat ggtgccaagc ttctctacgc atttgctgag 2100

gcaactgtac ccaaagtcac agtcatcacc aggaaggcct atggaggtgc ctatgatgtc 2160

atgagctcta agcacctttg tggtgatacc aactatgcct ggcccaccgc agagattgca 2220

gtcatgggag caaagggcgc tgtggagatc atcttcaaag ggcatgagaa tgtggaagct 2280

gctcaggcag agtacatcga gaagtttgcc aaccctttcc ctgcagcagt gcgagggttt 2340

gtggatgaca tcatccaacc ttcttccaca cgtgcccgaa tctgctgtga cctggatgtc 2400

ttggccagca agaaggtaca acgtccttgg agaaaacatg caaatattcc attgtaatct 2460

agagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 2520

tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 2580

agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 2640

gaggtgtggg aggtttttta gaggaacccc tagtgatgga gttggccact ccctctctgc 2700

gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc 2760

gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaa 2806

Claims (140)

1. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising, in 5 'to 3' order:

(a) a5 '-inverted terminal repeat (5' -ITR) sequence;

(b) a promoter sequence;

(c) a partial or complete coding sequence of PCCA or PCCB; and

(d)3 '-inverted terminal repeat (3' -ITR) sequence.

2. The rAAV of claim 1, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10, or hu 37.

3. The rAAV according to claim 2, wherein the AAV capsid is from AAV 9.

4. The rAAV according to claim 2, wherein the AAV capsid is from AAV 8.

5. The rAAV according to claim 1, wherein the AAV capsid is an AAV9 variant capsid.

6. The rAAV of any one of claims 1-5, wherein the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a Cytomegalovirus (CMV) immediate early gene promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, a CAG promoter, a PCCA gene-specific endogenous promoter, and a PCCB gene-specific endogenous promoter.

7. The rAAV of claim 6, wherein when part or the entire coding sequence in the vector genome is for PCCA, the promoter is a PCCA gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO: 34, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length.

8. The rAAV of claim 6, wherein when part or all of the coding sequence in the vector genome is for PCCB, the promoter is a PCCB gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO: 36, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length.

9. The rAAV of claim 6, wherein the promoter is a CBA promoter.

10. The rAAV according to any one of claims 1-9, wherein the 5 '-ITR and/or 3' -ITR sequences are from AAV 2.

11. The rAAV of claim 10, wherein the 5 '-ITR sequence and 3' -ITR sequence comprise SEQ ID NO: 15 or consist thereof.

12. The rAAV according to any one of claims 1-9, wherein the 5 '-ITR sequence and/or the 3' -ITR sequence is from a non-AAV 2 source.

13. The rAAV according to any one of claims 1-7 and 9-12, wherein part or all of the coding sequence of the PCCA is a wild-type coding sequence.

14. The rAAV of claim 13, wherein the coding sequence for PCCA comprises SEQ ID NO: 1.

15. the rAAV according to any one of claims 1-7 and 9-12, wherein part or all of the coding sequence of the PCCA is a codon-optimized coding sequence.

16. The rAAV of claim 15, wherein the coding sequence for PCCA comprises a sequence selected from SEQ ID NOs: 2-6.

17. The rAAV according to any one of claims 1-6 and 8-12, wherein part or all of the coding sequence of the PCCB is a wild-type coding sequence.

18. The rAAV of claim 17, wherein the coding sequence of the PCCB comprises SEQ ID NO: 7.

19. the rAAV according to any one of claims 1-6 and 8-12, wherein part or all of the coding sequence of the PCCB is a codon optimized coding sequence.

20. The rAAV of claim 19, wherein the coding sequence of the PCCB comprises a sequence selected from the group consisting of SEQ ID NOs: 8-12.

21. The rAAV according to any one of claims 1-7 and 9-16, wherein the packaged genome further comprises a truncated or complete nucleotide sequence of a human PCCA5 '-untranslated region (UTR) and/or 3' -UTR.

22. The rAAV of claim 21, wherein the complete nucleotide sequence of the human PCCA 5' -UTR comprises SEQ ID NO: 30.

23. the rAAV of claim 21, wherein the truncated nucleotide sequence of the human PCCA 5' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 30, and a nucleotide sequence of at least 100 contiguous nucleotides with at least 95% identity over a region of equal length.

24. The rAAV of claim 21, wherein the complete nucleotide sequence of the human PCCA 3' -UTR comprises SEQ ID NO: 31.

25. the rAAV of claim 21, wherein the truncated nucleotide sequence of the human PCCA 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 31, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

26. The rAAV according to any one of claims 1-6, 8-12, and 17-20, wherein the packaged genome further comprises a truncated or complete nucleotide sequence of a human PCCB5 '-untranslated region (UTR) and/or 3' -UTR.

27. The rAAV of claim 26, wherein the complete nucleotide sequence of the human PCCB 5' -UTR comprises the nucleotide sequence of SEQ ID NO: 32.

28. the rAAV of claim 26, wherein the truncated nucleotide sequence of the human PCCB 5' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 32, and a nucleotide sequence of at least 100 contiguous nucleotides that are at least 95% identical over a region of equal length.

29. The rAAV of claim 26, wherein the complete nucleotide sequence of the human PCCB 3' -UTR comprises SEQ ID NO: 33.

30. the rAAV of claim 26, wherein the truncated nucleotide sequence of the human PCCB 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 33, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

31. The rAAV according to any one of claims 1-30, wherein the packaged genome further comprises a polyadenylation signal sequence.

32. The rAAV of claim 31, wherein the polyadenylation signal sequence is selected from a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, a rabbit β globin polyadenylation signal sequence, a PCCA gene-specific endogenous polyadenylation signal sequence, a PCCB gene-specific endogenous polyadenylation signal sequence.

33. The rAAV of claim 32, wherein the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence or a SV40 polyadenylation signal sequence.

34. The rAAV of claim 33, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 22 or consist thereof.

35. The rAAV of claim 33, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO: 23 or consist thereof.

36. The rAAV according to any one of claims 1-7, 9-16, 21-25, and 31-32, wherein when part or all of the coding sequence in the vector genome is for PCCA, the polyadenylation signal sequence comprises a PCCA gene-specific endogenous polyadenylation signal sequence.

37. The rAAV of claim 36, wherein the PCCA gene-specific endogenous polyadenylation signal sequence comprises a sequence identical to SEQ ID NO: 35, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

38. The rAAV according to any one of claims 1-6, 8-12, 17-20, and 26-32, wherein when part or all of the coding sequence in the vector genome is for PCCB, the polyadenylation signal sequence comprises a PCCB gene-specific endogenous polyadenylation signal sequence.

39. The rAAV of claim 38, wherein the PCCB gene-specific endogenous polyadenylation signal sequence comprises a nucleotide sequence identical to SEQ ID NO: 37, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

40. The rAAV of any of claims 1-39, wherein the packaged genome further comprises one or more enhancer sequences.

41. The rAAV of claim 40, wherein the enhancer is selected from the group consisting of a Cytomegalovirus (CMV) immediate-early gene enhancer, a transthyretin enhancer (enTTR), a chicken β -actin (CBA) enhancer, an En34 enhancer, and an apolipoprotein E (ApoE) enhancer.

42. The rAAV of claim 41, wherein the enhancer is a CMV enhancer.

43. The rAAV of claim 42, wherein the enhancer comprises the amino acid sequence of SEQ ID NO: 19 or consist thereof.

44. The rAAV according to claims 40-43, wherein the enhancer is upstream of the promoter sequence.

45. The rAAV of any of claims 1-44, wherein the packaged genome further comprises one or more intron sequences.

46. The rAAV of claim 45, wherein the intron is selected from the group consisting of the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, and the hFIX intron.

47. The rAAV of claim 46, wherein the intron is the SV40 small T intron or the rHBB intron.

48. The rAAV of claim 47, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 20 or consist thereof.

49. The rAAV of claim 47, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 21 or consist thereof.

50. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and packaged therein a vector genome comprising as operably linked components in 5 'to 3' order:

(a) 5' -ITR sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) a partial or complete coding sequence of PCCA or PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' -ITR sequence.

51. The rAAV of claim 50, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10, or hu 37.

52. The rAAV of claim 51, wherein the AAV capsid is from AAV8 or AAV 9.

53. The rAAV according to claim 50, wherein the AAV capsid is an AAV9 variant capsid.

54. The rAAV of any one of claims 50-53, wherein the promoter is selected from the group consisting of a chicken β -actin (CBA) promoter, a Cytomegalovirus (CMV) immediate early gene promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, an α -1 antitrypsin factor (A1AT) promoter, a CAG promoter, a PCCA gene-specific endogenous promoter, and a PCCB gene-specific endogenous promoter.

55. The rAAV of claim 54, wherein when part or the entire coding sequence in the vector genome is for PCCA, the promoter is a PCCA gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO: 34, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length.

56. The rAAV of claim 54, wherein when part or the entire coding sequence in the vector genome is for PCCB, the promoter is a PCCB gene-specific endogenous promoter comprising a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO: 36, and a nucleotide sequence of at least 15 contiguous nucleotides with at least 95% identity over a region of equal length.

57. The rAAV of claim 54, wherein the promoter is a CBA promoter.

58. The rAAV of any one of claims 50-57, wherein the 5 '-ITR and/or 3' -ITR sequences are from AAV 2.

59. The rAAV of claim 58, wherein the 5 '-ITR sequence and 3' -ITR sequence comprise SEQ ID NO: 15 or consist thereof.

60. The rAAV of any one of claims 50-57, wherein the 5 '-ITR sequence and/or the 3' -ITR sequence is from a non-AAV 2 source.

61. The rAAV of any one of claims 50-55 and 57-60, wherein part or all of the coding sequence for the PCCA is a wild-type coding sequence.

62. The rAAV of claim 61, wherein the coding sequence for PCCA comprises the amino acid sequence of SEQ ID NO: 1.

63. the rAAV of any one of claims 50-55 and 57-60, wherein part or all of the coding sequence for the PCCA is codon-optimized coding sequence.

64. The rAAV of claim 63, wherein the coding sequence for PCCA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-6.

65. The rAAV of any one of claims 50-54 and 56-60, wherein part or all of the coding sequence of the PCCB is wild-type coding sequence.

66. The rAAV of claim 65, wherein the coding sequence for the PCCB comprises the amino acid sequence of SEQ ID NO: 7.

67. the rAAV of any one of claims 50-54 and 56-60, wherein part or all of the coding sequence of the PCCB is codon-optimized coding sequence.

68. The rAAV of claim 67, wherein the coding sequence for the PCCB comprises a sequence selected from the group consisting of SEQ ID NOs: 8-12.

69. The rAAV of any one of claims 50-68, wherein the polyadenylation signal sequence is selected from a Bovine Growth Hormone (BGH) polyadenylation signal sequence, a SV40 polyadenylation signal sequence, a rabbit β globin polyadenylation signal sequence, a PCCA gene-specific endogenous polyadenylation signal sequence, a PCCB gene-specific endogenous polyadenylation signal sequence.

70. The rAAV of claim 69, wherein the polyadenylation signal sequence is a Bovine Growth Hormone (BGH) polyadenylation signal sequence or a SV40 polyadenylation signal sequence.

71. The rAAV of claim 70, wherein the polyadenylation signal sequence comprises the amino acid sequence of SEQ ID NO: 22 or consist thereof.

72. The rAAV of claim 70, wherein the polyadenylation signal sequence comprises the amino acid sequence of SEQ ID NO: 23 or consist thereof.

73. The rAAV of any one of claims 50-55, 57-64, and 69, wherein the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence when part or all of the coding sequence in the vector genome is for PCCA.

74. The rAAV of claim 73, wherein the PCCA gene-specific endogenous polyadenylation signal sequence comprises an amino acid sequence identical to SEQ ID NO: 35, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

75. The rAAV according to any one of claims 50-54, 56-60, and 65-69, wherein when part or all of the coding sequence in the vector genome is for PCCB, the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence.

76. The rAAV of claim 75, wherein the PCCB gene-specific endogenous polyadenylation signal sequence comprises a nucleotide sequence identical to SEQ ID NO: 37, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

77. The rAAV according to any one of claims 50-76, wherein the enhancer is selected from the group consisting of a cytomegalovirus immediate early gene (CMV) enhancer, a transthyretin enhancer (enTTR), a chicken β -actin (CBA) enhancer, an En34 enhancer, and an ApoE enhancer.

78. The rAAV according to claim 77, wherein the enhancer is a CMV enhancer.

79. The rAAV of any one of claims 50-78, wherein the intron is selected from the SV40 small T intron, the rabbit hemoglobin subunit beta (rHBB) intron, the human beta globin IVS2 intron, the Promega chimeric intron, or the hFIX intron.

80. The rAAV of claim 79, wherein the intron is the SV40 small T intron or the rHBB intron.

81. The rAAV according to any one of claims 50-55, 57-64, 69-74, and 77-80, wherein when part or all of the coding sequence in the vector genome is for PCCA, the packaged genome further comprises a truncated or all of a nucleotide sequence of the human PCCA 5' -untranslated region (UTR) located between vector genome elements (d) and (e).

82. The rAAV of claim 81, wherein the entire nucleotide sequence of the human PCCA 5' -UTR comprises the nucleotide sequence of SEQ ID NO: 30.

83. the rAAV of claim 81, wherein the truncated nucleotide sequence of the human PCCA 5' -UTR comprises a nucleotide sequence that is identical to SEQ ID NO: 30, and a nucleotide sequence of at least 100 contiguous nucleotides with at least 95% identity over a region of equal length.

84. The rAAV according to any one of claims 50-54, 56-60, 65-72, and 75-80, wherein when part or all of the coding sequence in the vector genome is for PCCB, the packaged genome further comprises a truncated or all of a nucleotide sequence of the human PCCB 5' -untranslated region (UTR) located between vector genomic elements (d) and (e).

85. The rAAV of claim 84, wherein the complete nucleotide sequence of the human PCCB 5' -UTR comprises the nucleotide sequence of SEQ ID NO: 32.

86. the rAAV of claim 84, wherein the truncated nucleotide sequence of the human PCCB 5' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 32, and a nucleotide sequence of at least 100 contiguous nucleotides that are at least 95% identical over a region of equal length.

87. The rAAV according to any one of claims 50-55, 57-64, 69-74, and 77-83, wherein when part or all of the coding sequence in the vector genome is for PCCA, the packaged genome further comprises a truncated or all of a nucleotide sequence of the human PCCA 3' -untranslated region (UTR) located between vector genome elements (f) and (g).

88. The rAAV of claim 87, wherein the entire nucleotide sequence of the human PCCA 3' -UTR comprises SEQ ID NO: 31.

89. the rAAV of claim 87, wherein the truncated nucleotide sequence of the human PCCA 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 31, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

90. The rAAV according to any one of claims 50-54, 56-60, 65-72, 75-80, and 84-86, wherein when part or all of the coding sequence in the vector genome is for PCCB, the packaged genome further comprises a truncated or all nucleotide sequence of the human PCCB 3' -untranslated region (UTR) located between vector genomic elements (f) and (g).

91. The rAAV of claim 90, wherein the complete nucleotide sequence of the human PCCB 3' -UTR comprises SEQ ID NO: 33.

92. the rAAV of claim 90, wherein the truncated nucleotide sequence of the human PCCB 3' -UTR comprises a nucleotide sequence identical to SEQ ID NO: 33, and a nucleotide sequence of at least 15 contiguous nucleotides that are at least 95% identical over a region of equal length.

93. The rAAV of any one of claims 50-92, wherein the vector genome comprises a consensus Kozak sequence upstream of genomic element (e).

94. The rAAV of claim 93, wherein the consensus Kozak sequence comprises SEQ ID NO: 24.

95. a recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' -Inverted Terminal Repeat (ITR) sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 1-6 or a coding sequence for PCCA selected from SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' -Inverted Terminal Repeat (ITR) sequences.

96. The rAAV of claim 95, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, rh10, or hu 37.

97. The rAAV according to claim 96, wherein the AAV capsid is from AAV 9.

98. The rAAV according to claim 96, wherein the AAV capsid is from AAV 8.

99. The rAAV according to claim 95, wherein the AAV capsid is an AAV9 variant capsid.

100. A recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) a 5' -Inverted Terminal Repeat (ITR) sequence;

(b) an enhancer sequence;

(c) a promoter sequence;

(d) an intron sequence;

(e) selected from the group consisting of SEQ ID NOs: 1-6 or a coding sequence for PCCA selected from SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a polyadenylation signal sequence; and

(g) 3' -Inverted Terminal Repeat (ITR) sequences.

101. A recombinant adeno-associated virus (rAAV) useful in the treatment of Propionemia (PA), the rAAV comprising an AAV9 capsid and a vector genome packaged therein, the vector genome comprising, as operably linked components, in a5 'to 3' order:

(a) AAV 25' -Inverted Terminal Repeat (ITR) sequence;

(b) a CMV enhancer sequence;

(c) a CBA promoter sequence;

(d) rHBB or SV40 small T intron sequences;

(e) selected from the group consisting of SEQ ID NOs: 1-6 or a coding sequence for PCCA selected from SEQ ID NOs: 7-12, the coding sequence of PCCB;

(f) a BGH or SV40 polyadenylation signal sequence; and

(g) AAV 23' -Inverted Terminal Repeat (ITR) sequences.

102. The rAAV of any one of claims 95-101, wherein when the coding sequence in the vector genome is for PCCA, the packaged genome further comprises a truncated or complete nucleotide sequence of the human PCCA 5' -UTR located between vector genome elements (d) and (e).

103. The rAAV according to any one of claims 95-101, wherein when the coding sequence in the vector genome is for PCCB, the packaged genome further comprises a truncated or complete nucleotide sequence of the human PCCB 5' -UTR located between vector genomic elements (d) and (e).

104. The rAAV of any one of claims 95-102, wherein when the coding sequence in the vector genome is for PCCA, the packaged genome further comprises a truncated or complete nucleotide sequence of the human PCCA 3' -UTR located between vector genome elements (f) and (g).

105. The rAAV according to any one of claims 95-101 and 103, wherein when the coding sequence in the vector genome is for PCCB, the packaged genome further comprises a truncated or complete nucleotide sequence of the human PCCB 3' -UTR located between vector genomic elements (f) and (g).

106. The rAAV of any one of claims 95-105, wherein the vector genome comprises a consensus Kozak sequence upstream of genomic element (e).

107. The rAAV of claim 106, wherein the consensus Kozak sequence comprises SEQ ID NO: 24.

108. a composition comprising the rAAV of any one of the preceding claims and a pharmaceutically acceptable carrier.

109. A method of treating Propionemia (PA) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of the rAAV of any one of claims 1-107 or the composition of claim 108.

110. A method of treating Propionemia (PA) in a human subject, the method comprising administering to the human subject:

1. a therapeutically effective amount of a composition comprising the rAAV of any one of claims 1-7, 9-16, 21-25, 31-37, 40-55, 57-64, 69-74, 77-83, 87-89, 93-102, 104, and 106-107 in which the coding sequence is directed to PCCA and not PCCB, the promoter is a PCCA gene-specific endogenous promoter and not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence and not a PCCB gene-specific endogenous polyadenylation signal sequence; and/or

2. A therapeutically effective amount of a composition comprising the rAAV of any one of claims 1-6, 8-12, 17-20, 26-35, 38-54, 56-60, 65-72, 75-80, 84-86, 90-101, 103, and 105-107 in which the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene-specific endogenous promoter and not a PCCA gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene-specific endogenous polyadenylation signal sequence and not a PCCA gene-specific endogenous polyadenylation signal sequence.

111. The method of claim 110, wherein the compositions of (1) and (2) are administered simultaneously, sequentially, or separately.

112. A method of treating Propionemia (PA) in a human subject diagnosed as having at least one mutation in PCCA, the method comprising administering to the human subject a therapeutically effective amount of a composition comprising the rAAV of any one of claims 1-7, 9-16, 21-25, 31-37, 40-55, 57-64, 69-74, 77-83, 87-89, 93-102, 104, and 106, in which the coding sequence is directed to PCCA rather than to PCCB, the promoter is a PCCA gene specific endogenous promoter and not a PCCB gene specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence and not a PCCB gene-specific endogenous polyadenylation signal sequence.

113. The method of claim 112, wherein the mutation in PCCA is selected from table 1.

114. A method of treating Propionemia (PA) in a human subject diagnosed as having at least one mutation in PCCB, the method comprising administering to the human subject a therapeutically effective amount of a composition comprising the rAAV of any one of claims 1-6, 8-12, 17-20, 26-35, 38-54, 56-60, 65-72, 75-80, 84-86, 90-101, 103, and 105-107, in which the coding sequence is directed to PCCB and not to PCCA, the promoter is a PCCB gene specific endogenous promoter and not a PCCA gene specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene specific endogenous polyadenylation signal sequence and not a PCCA gene specific endogenous polyadenylation signal sequence.

115. The method of claim 114, wherein the mutations in the PCCB species are selected from table 2.

116. The method of any one of claims 109-115, wherein the rAAV or the composition is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, or intravenously.

117. The method of claim 116, wherein the rAAV or the composition is administered intravenously.

118. The method of any one of claims 109-117, wherein the rAAV is at about 1x10¹¹To about 1x10¹⁴Genomic Copy (GC)/kg.

119. The method of claim 118, wherein the rAAV is administered at about 1x10¹²To about 1x10¹³Genomic Copy (GC)/kg.

120. The method of any one of claims 116-119, wherein administering the rAAV comprises administering:

a.) a single dose of rAAV; or

b.) multiple doses of rAAV.

121. An isolated nucleic acid encoding SEQ ID NO: 16, wherein the nucleic acid sequence is identical to the PCCA polypeptide of SEQ ID NO: 1 has less than 80% identity to the wild type coding sequence.

122. The recombinant nucleic acid of claim 121, wherein the nucleic acid sequence comprises a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 80% identical.

123. The recombinant nucleic acid of claim 122, wherein the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2-6.

124. An isolated nucleic acid encoding SEQ ID NO: 17, wherein the nucleic acid sequence is identical to the PCCB polypeptide of SEQ ID NO: 7 has less than 80% identity to the wild type coding sequence.

125. The recombinant nucleic acid of claim 124, wherein the nucleic acid sequence comprises a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8-12, which is at least 80% identical.

126. The recombinant nucleic acid of claim 125, wherein the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 8-12.

127. A recombinant nucleic acid comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 80% identical.

128. The recombinant nucleic acid of claim 127, wherein the recombinant nucleic acid comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 90% identical.

129. The recombinant nucleic acid of claim 128, wherein said recombinant nucleic acid comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 2-6, which is at least 95% identical.

130. A recombinant nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 2-6.

131. A recombinant nucleic acid comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8-12, which is at least 80% identical.

132. The recombinant nucleic acid of claim 131, wherein the recombinant nucleic acid comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 8-12, which is at least 90% identical.

133. The recombinant nucleic acid of claim 132, wherein the recombinant nucleic acid comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 8-12, which is at least 95% identical.

134. A recombinant nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 8-12.

135. The recombinant nucleic acid of any one of claims 121-134, wherein the nucleic acid sequence further comprises at the 3' end one or more stop codons selected from the group consisting of TGA, TAA and TAG.

136. A rAAV comprising the recombinant nucleic acid of any one of claims 121-135.

137. A host cell comprising the recombinant nucleic acid of any one of claims 121-135 or the rAAV of claim 136.

138. The host cell of claim 137, wherein the host cell is selected from the group consisting of HeLa, Cos-7, HEK293, a549, BHK, Vero, RD, HT-1080, ARPE-19, or MRC-5 cells.

139. The rAAV of any one of claims 1-7, 9-16, 21-25, 31-37, 40-55, 57-64, 69-74, 77-83, 87-89, 93-102, 104, and 106-107, wherein the coding sequence is for PCCA and not PCCB, the promoter is a PCCA gene-specific endogenous promoter and not a PCCB gene-specific endogenous promoter, and the polyadenylation signal sequence is a PCCA gene-specific endogenous polyadenylation signal sequence and not a PCCB gene-specific endogenous polyadenylation signal sequence.

140. The rAAV of any one of claims 1-6, 8-12, 17-20, 26-35, 38-54, 56-60, 65-72, 75-80, 84-86, 90-101, 103, and 105-107, wherein the coding sequence is for PCCB and not PCCA, the promoter is a PCCB gene specific endogenous promoter and not a PCCA gene specific endogenous promoter, and the polyadenylation signal sequence is a PCCB gene specific endogenous polyadenylation signal sequence and not a PCCA gene specific endogenous polyadenylation signal sequence.

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